A total of 56 strains of Lactobacillus were isolated using MRS medium from a sea cucumber (Apostichopus japonicus) culture pond in Dongying, Shandong Province. Their antibacterial activities were analyzed using the Oxford cup method, and Vibrio splendidus and Pseudoalteromonas nigrifaciens were used as indicator bacteria. A Lactobacillus strain, CSND-6, showed strong antibacterial activity. The morphology, antibacterial activities of intracellular and extracellular components, physiological and biochemical tests, 16S rDNA sequence analysis, growth characteristics, and effects of strain CSND-6 on sea cucumbers were studied. The results showed that strain CSND-6 had a significant inhibitory effect on the growth of V. splendens and P. nigrifaciens. The inhibition zones of the intracellular components were 17 mm and 21 mm, and that of extracellular components were 25 mm and 33 mm for V. splendens and P. nigrifaciens, respectively. The safety analysis showed that the strain was safe for sea cucumbers. The results of the physiological and biochemical tests and 16S rDNA sequence analysis showed that the strain CSND-6 belongs to family Lactobacillaceae; the similarity between CSND-6 and Lactobacillus paracasei JCM1171 was 99.83%. The strain CSND-6 grew rapidly between 30℃ to 44℃ and in a pH between 6 to 8. It entered the logarithmic growth phase after 24 h and reached the highest concentration (2.10×109 CFU/ml) after 28~32 h. Since strain CSND-6 was isolated from the environment of the sea cucumber culture ponds and exerted strong inhibitory effects on the main pathogenic bacteria of sea cucumbers, it holds potential as an agent to prevent and control infections and diseases in sea cucumber. This study provides the foundation for the development and utilization of probiotics in sea cucumber cultures.