Objective To establish a Raw-264 cell model for evaluating virulence of Vibrio parahaemolyticus. Methods The CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit was employed to test the Vibrio parahaemolyticus-induced cytotoxicity against Raw264 cells. Results The protocols were optimized as follows: Vibrio parahaemolyticus cells and Raw264 cells were incubated for 1.5 h at 37℃ with a MOI of 5; the mixture was then pelleted, and the 10-fold diluted supernatant was incubated with the substrate for 10 min. The cytotoxicity against Raw-264 cells determined for the opaR mutant was significantly higher than that for the wild-type strain. Conclusions A Raw-264 cellular model was established for evaluating virulence of Vibrio parahaemolyticus.