Marine microorganisms, though microscopic, represent a vast reservoir of organic carbon and play a central role in element cycling and energy flow within marine ecosystems. As key contributors to the biogeochemical cycling of carbon, these microorganisms mediate essential processes, including carbon fixation, methane metabolism, and carbon degradation. Carbon fixation, where microorganisms convert CO₂ into organic carbon, is vital for understanding marine carbon sequestration potential. Six primary microbial carbon fixation pathways have been identified: Calvin-Benson-Bassham cycle (CBB), reductive acetyl-CoA (Wood-Ljungdahl) pathway (WL), reductive tricarboxylic acid cycle, 3-hydroxypropionate bicycle (3HB), 3-hydroxypropionate/4-hydroxybutyrate cycle (3HB/4HB), and dicarboxylate/4-hydroxybutyrate cycle (DC/4HB). Among these, the Calvin cycle dominates in phototrophic autotrophs, requiring high oxygen levels and contributing significantly to global CO₂ regulation via photosynthesis. By contrast, the 3-HB and WL pathways thrive in low-oxygen and anaerobic conditions, respectively, showing high carbon fixation efficiency, particularly in extremophilic communities. With advances in metagenomics, research on marine microorganisms in complex ecosystems such as the Beibu Gulf (Northern Bay of Guangxi) has deepened, offering valuable insights into microbial communities and their roles in carbon cycling. The Beibu Gulf is strategically important ecologically and economically, lying at the intersection of South China, Southwest China, and ASEAN economic zones. However, the carbon sink potential of its marine microbial communities remains largely unexplored, and the factors influencing this potential are not yet fully understood. Thus, this study aims to evaluate the carbon fixation potential of marine microorganisms in the Beibu Gulf and identify the environmental factors shaping microbial community structure. Using field data and metagenomic sequencing, we conducted seasonal sampling at 40 stations in the bay, coastal, and island regions during the wet season (October 2022) and dry season (January 2023). By analyzing water physicochemical parameters and the abundance of microbial carbon fixation pathways, we examined the spatiotemporal distribution of these pathways across regions and seasons and their correlation with environmental variables. The study reveals significant spatiotemporal variations in the abundance of microbial carbon fixation genes. During the wet season, carbon fixation primarily occurred via the CBB and 3HB pathways, with key genes such as meh and mcl showing notable regional variability in abundance, and K14469 was detected exclusively in the bay. During the dry season, prKB, meh, mct, and mcl exhibited significant regional variability. Notably, WL pathway genes cooS and acsE, which are crucial for carbon fixation under anaerobic conditions, were detected exclusively in island areas during the dry season. Principal coordinate analysis results demonstrated distinct regional differences in carbon sink gene composition across seasons, with the most prominent variance occurring between bay and island areas. Similarity analysis indicated significant differences in carbon fixation gene composition between regions during the wet (r=0.111, P=0.035) and dry seasons (r=0.416, P=0.001), with bay-island differences particularly pronounced. Seasonal analysis of the microbial carbon fixation pathways showed that the CBB pathway was dominant during the wet season, accounting for approximately 70% of the total carbon fixation pathways detected. By contrast, during the dry season, the relative abundance of the CBB and 3HB pathways was nearly equivalent, representing 49% and 50% of the total pathways, respectively. Furthermore, metagenomic data suggested that the overall carbon fixation potential of marine microorganisms in the Beibu Gulf was significantly higher during the wet season, with total pathway abundance ranging from 7.93 TPM to 55.39 TPM, averaging 23.68 TPM, which is 1.75 times greater than that during the dry season. In addition, spatial distribution analysis indicated that, during the wet season, the CBB pathway had a substantially higher relative abundance (4.96–48.43 TPM, averaging 16.58 TPM) than the 3HB pathway (2.97–14.51 TPM, averaging 7.10 TPM). The abundance of carbon fixation pathways significantly correlated with multiple environmental variables. The CBB pathway was highly correlated with factors such as nitrate (NO₃-N), nitrite (NO₂-N), salinity (SAL), total nitrogen (TN), dissolved inorganic phosphate (DIP), water temperature (WT), total organic carbon (TOC), and pH. For the 3HB pathway, WT and dissolved oxygen (DO) were significantly correlated. Overall, the total carbon fixation potential was predominantly influenced by SAL, DIP, NO₂-N, NO₃-N, TN, and TOC, indicating these variables as the primary drivers impacting microbial carbon sink potential in the Beibu Gulf. In summary, this study provides a comprehensive assessment of the carbon fixation potential of marine microorganisms in the Beibu Gulf and identifies key environmental factors influencing carbon sink capacity. The findings underscore the substantial role of environmental conditions in shaping the carbon fixation pathways across different seasons and regions, highlighting the complex and dynamic microbial carbon cycling processes in the Beibu Gulf. This study contributes valuable insights into marine microbial carbon sequestration and offers a scientific basis for future studies on the ecological impact of microbial communities in marine carbon cycling.