Nitrite is strongly toxic to shrimp, and is one of the main environmental stress factors affecting the growth and survival of shrimp. High nitrite levels in aquaculture systems are often associated with the phenomenon of shrimp exoskeleton softening (soft-shell), and the mechanism underlying this must be further studied. In order to reveal the mechanism of nitrite resulting in the exoskeleton softening of Penaeus vannamei, this study chosen shrimp from three distinct groups: hard-shell shrimp (CH) randomly sampled from three ponds with nitrite concentration of (4.35±1.07) mg/L as the control group, and both hard-shell shrimp (NH) and soft-shell shrimp (NS) collected from three other ponds with nitrite concentration of (21.05±0.84) mg/L, as experimental groups. The physiological indexes of the hepatopancreas were determined, and transcriptomic analysis was performed. An average of 45,457,339, 43,648,589, and 44,293,378 clean reads were obtained in the CH, NS, and NH groups, respectively. The obtained unigenes were annotated in the NR (NCBI non-redundant protein sequences), NT (NCBI nucleotide sequences), PFAM (protein family), KOG (euKaryotic Ortholog Groups), SwissProt (a manually annotated and reviewed protein sequence database), GO (Gene Ontology), and KO (KEGG Orthology) databases. The majority of the genes were annotated in the NR database (92.83%), followed by the EggNOG (58.58%), Pfam (57.39%), Swiss-Prot (53.82%), GO (52.49%), and KEGG (43.22%) databases. Overall, 28,878 out of 30,746 genes (accounting for 93.92% of the total) were annotated in at least one database. The transcriptomic results showed that a total of 3,284 differentially expressed genes (DEGs) were identified across different groups. Among these, the NS vs. CH group had the highest number of DEGs, at 2,924, including 1,375 up-regulated genes and 1,549 down-regulated genes. The NH vs. CH group had the fewest DEGs, with 479, including 313 up-regulated genes and 166 down-regulated genes. In the NS vs. NH group, 837 DEGs were identified, with 588 up-regulated genes and 249 down-regulated genes. According to the KEGG enrichment results, the down-regulated differentially expressed genes in the NS vs. NH group were significantly enriched in the retinol metabolism pathway (ko00830) and the cytochrome P450 metabolism of xenobiotics pathway (ko00980). In the NS vs. CH group, the most significantly down-regulated pathway was also the cytochrome P450 metabolism of xenobiotics pathway (ko00980), with significant enrichment observed in the pentose and glucuronate interconversions pathway (ko00040), pancreatic secretion pathway (ko04972), and retinol metabolism pathway (ko00830). In the NH vs. CH group, the aldosterone-regulated sodium reabsorption pathway (ko04960) and the progesterone-mediated oocyte maturation pathway (ko04914) were significantly enriched. Additionally, the PPAR signaling pathway (ko03320) and the fatty acid degradation pathway (ko00071) were significantly enriched in both the NS vs. NH group and the NS vs. CH group. The results showed that high nitrite concentrations significantly inhibited the expression levels of glucose-6-phosphate isomerase, trehalose transporter, UDP-N-acetylglucosamine pyrophosphorylase, glutamine synthetase, and other key genes related to chitin synthesis. In addition, the expression levels of genes related to chitin degradation (chitinase, chitotriase, neutral α-glucosidase) in the NS group were significantly increased. According to the results of GO enrichment analysis, under nitrite stress, hydrolase in the NS group, hydrolyzed O-glycosylated compounds (GO: 0004553), hydrolase, acting on glycosylated bond (GO: 0016798) related gene expression were upregulated, which may lead to a decrease in cuticle chitin content. In addition, GO enrichment analysis showed that, compared with the CH group, NS differentially expressed genes in pre-ribosomes (GO: 0030684), nucleolus (GO: 0005730), cell modification of amino acid metabolism (GO: 0006575), rRNA metabolism (GO: 0016072), and rRNA processing (GO: 0016072). Significant enrichment, such as that observed for long alcohol diphosphate oligosaccharide glycosyltransferase (RPN), translation initiation factor 2 (EIF2), may lead to stratum corneum disorganization. In addition, genes related to mineral absorption (calintegrin binding protein, high affinity copper uptake protein, ferritin, etc.) and calreticulin genes were significantly down-regulated in the NS group, which may directly affect exoskeleton sclerosis. KEGG enrichment analysis showed that, compared with the other two groups, the differentially expressed genes in the NS group were significantly enriched in the PPAR signaling pathway (ko03320) and the fatty acid degradation pathway (ko00071). Specifically, the genes related to lipid metabolism were down-regulated in the NS group, which may lead to the absence of extra energy for exoskeleton hardening. Physiological indexes demonstrated that the activities of chitinase and β-N-acetylglucosaminase in the NS group were significantly higher than those in the NH group and the CH group (P＜0.05). Conversely, trehalase activity in the CH group surpassed that of the NS group, while Ca²⁺-ATPase activity was notably higher in the CH group compared to the NH group (P＜0.05). The content of Ca²⁺ in the hepatopancreas in the NH group was significantly higher than that in the NS and CH groups (P＜0.05). These results indicate that high nitrite levels in water may affect exoskeleton hardness of P. vannamei through a comprehensive mechanism, including reduction of chitin content, damaging cuticle conformation, and reducing mineral deposition. This study provides a theoretical basis for further exploring the effect of nitrite on shrimp exoskeleton.