Abstract: Vibrio parahaemolyticus is a foodborne pathogen found widely in aquatic products, the infectious outbreaks of which are often associated with serious food safety issues, including food poisoning. In order to determine whether the regulatory factor RpoS played a key role in the resistance of V. parahaemolyticus to environmental stresses, homologous recombination technology was used to generate rpoS gene deletion strain ATCC33846/ΔrpoS and rpoS gene backfill strain ΔrpoS/pBAD33T-rpoS in this study. The results revealed that the growth of ATCC33846/ΔrpoS was significantly inhibited in response to exposure to different temperature, NaCl concentration, oligotrophic, and pH conditions, and that there were reductions in the mobility and biofilm formation capacity of this strain. Scanning electron microscopy revealed alterations in the surface morphology of ATCC33846/ΔrpoS cell membranes, characterized by the presence of obvious folds. In addition, Fourier transform infrared spectroscopy analysis indicated a weakening of the hydrogen bonds of ATCC33846/ΔrpoS, thereby influencing the lipid content of cell membranes, and causing reductions in the extracellular secretion of polysaccharides. Moreover, in response to treatment with norfloxacin, polymyxin B, enrofloxacin, chloramphenicol, and rifampicin, significant concentration-dependent reductions in the antibiotic resistance of ATCC33846/ΔrpoS were detected. These findings provide evidence to indicate that RpoS is a key regulator of the resistance of V. parahaemolyticus to external environmental stress.