Surfactin exhibits multifunctional properties, including antimicrobial, anti-inflammatory, and antitumor activities, positioning it as a promising novel food additive. However, its specific effects on the gut environment remain largely unexplored. To investigate this, we conducted in vitro fermentation simulations to assess the impact of surfactin on gut microbiota. Additionally, we evaluated surfactin's inhibitory effects on HCT-116 cells. The results of the fermentation simulation indicated that surfactin significantly increased butyrate production and enhanced the relative abundance of Megamonas, Alistipes, the Oscillospiraceae NK4A214 group, and Methanobrevibacter smithii, while markedly inhibiting Desulfobacterota, potentially facilitating amino acid and lipid metabolism. Furthermore, surfactin significantly inhibited the proliferation of HCT-116 cells and promoted their apoptosis, without exhibiting cytotoxicity towards normal human intestinal epithelial crypt (HIEC) cells. In summary, surfactin demonstrates a favorable safety profile in the intestinal environment. This study contributes to our understanding of the interaction between surfactin and the gut environment.