The study evaluates the in vitro safety and functional properties of the novel Bacillus coagulans strain CGI314. The strain was tested for its ability to survive extreme temperatures (40°C, 70°C, 90°C), gastric acid (pH 3.0), and bile salts (0.3%), demonstrating strong survival under gastrointestinal conditions. Spores and vegetative cells adhered to mucous-producing intestinal cell lines, showing moderate auto-aggregation and the ability to co-aggregate with pathogenic bacteria. The strain exhibited broad antagonistic activity against various pathogens, including those in the urinary tract, intestinal, oral, and skin systems. Metabolomic profiling revealed the strain's ability to synthesize amino acids, vitamins, and short-chain fatty acids. It also demonstrated strong antioxidant capacity, with cytoprotective effects in HepG2 and HT-29 cell lines. The strain significantly increased the expression of tight junction proteins and partially ameliorated the damage caused by hydrogen peroxide-induced disruption of the intestinal-epithelial barrier. The study also assessed the strain's ability to reduce cholesterol, produce lactic acid, and exhibit anti-inflammatory activity. Overall, these findings suggest that B. coagulans CGI314 has promising probiotic potential for use in food products. The strain showed strong resistance to simulated gastric and intestinal conditions, outperforming conventional probiotic strains. It also demonstrated low hemolytic activity and no significant cytotoxicity in HT-29 and HepG2 cell lines. The strain exhibited strong antioxidant capacity, comparable to that of the positive control, vitamin C. Additionally, it showed the ability to synthesize amino acids and vitamins, and produce short-chain fatty acids. The strain also demonstrated the ability to ferment various carbohydrates and exhibited a broad enzymatic profile. These findings indicate that B. coagulans CGI314 is a promising probiotic candidate with potential applications in functional foods.The study evaluates the in vitro safety and functional properties of the novel Bacillus coagulans strain CGI314. The strain was tested for its ability to survive extreme temperatures (40°C, 70°C, 90°C), gastric acid (pH 3.0), and bile salts (0.3%), demonstrating strong survival under gastrointestinal conditions. Spores and vegetative cells adhered to mucous-producing intestinal cell lines, showing moderate auto-aggregation and the ability to co-aggregate with pathogenic bacteria. The strain exhibited broad antagonistic activity against various pathogens, including those in the urinary tract, intestinal, oral, and skin systems. Metabolomic profiling revealed the strain's ability to synthesize amino acids, vitamins, and short-chain fatty acids. It also demonstrated strong antioxidant capacity, with cytoprotective effects in HepG2 and HT-29 cell lines. The strain significantly increased the expression of tight junction proteins and partially ameliorated the damage caused by hydrogen peroxide-induced disruption of the intestinal-epithelial barrier. The study also assessed the strain's ability to reduce cholesterol, produce lactic acid, and exhibit anti-inflammatory activity. Overall, these findings suggest that B. coagulans CGI314 has promising probiotic potential for use in food products. The strain showed strong resistance to simulated gastric and intestinal conditions, outperforming conventional probiotic strains. It also demonstrated low hemolytic activity and no significant cytotoxicity in HT-29 and HepG2 cell lines. The strain exhibited strong antioxidant capacity, comparable to that of the positive control, vitamin C. Additionally, it showed the ability to synthesize amino acids and vitamins, and produce short-chain fatty acids. The strain also demonstrated the ability to ferment various carbohydrates and exhibited a broad enzymatic profile. These findings indicate that B. coagulans CGI314 is a promising probiotic candidate with potential applications in functional foods.