Bile salts are crucial microbe-selective inhibitors present in the intestinal tracts of humans and other animals. Environmental and clinical strains of Vibrio parahaemolyticus (V. parahaemolyticus) exhibited different biofilm-forming abilities under bile salt incubation. In order to find an effective way to eliminate biofilm, in this study, environmental strains were subjected to mouse gastrointestinal (GI) stimulation and cultured in medium containing 0.06 % bile salts. The effects of GI stimulation on V. parahaemolyticus biofilm formation were evaluated by biofilm cells assay, atomic force microscopy (AFM) assay, confocal laser scanning microscopy (CLSM) assay, extracellular polysaccharide (EPS) assay, and salmon surface biofilm formation assay. The results showed that GI stimulation diminished the ability of V. parahaemolyticus to form biofilm, significantly reduced biofilm cells, decreased the level of EPS, and destroyed the biofilm structure. For the biofilm formed by V. parahaemolyticus after GI stimulation, AFM observed that the appearance of the biofilm became inhomogeneous and rough, and CLSM observed that the 3D structure of the biofilm became dispersed and sparse. GI stimulation reduced the ability of V. parahaemolyticus to form biofilms on the surface of salmon containing 0.06 % bile salts at both 12 h and 24 h, as evidenced by a decrease in the number of adherent cells. Comparing biofilms formed by tdh-positive V. parahaemolyticus before and after undergoing GI stimulation, a total of 1169 differentially expressed genes (DEGs) were identified by RNA sequencing. And 10 of the biofilm-related genes displayed significant down-regulation after GI stimulation. Enrichment analysis of DEGs revealed that affecting the switch between succinate and fumarate in the TCA cycle could inhibit biofilm formation. This study offers new insights into strategies for preventing biofilm formation by foodborne pathogens.