Pathogenic (ATCC 33847: tdh+/trh-/tlh+; ATCC 17802: tdh-/trh+/tlh+) and non-pathogenic (G2: tdh-/trh-/tlh+; G8: tdh-/trh-/tlh+) V. parahaemolyticuss strains were mixed at 9∶1 and 1∶1 ratios and cultured at 37°C in tryptic soy broth (TSB) or cooked shrimp for 10 h. The rate of isolation of the pathogenic strains after 10 h was determined by colony hybridization and PCR method, and the results showed that the isolation rate of the pathogenic strains decreased after co-culturing in TSB and cooked shrimp. The isolation rate of the pathogenic strains was more prominent in the shrimp samples, and when the strain ATCC 17802 was mixed with strain G8 in a 9∶1 ratio in the shrimp samples, no pathogenic strain was detected after 10 h. Negative effects of mixed culture on the growth of pathogenic V. parahaemolyticus are one of reasons for the rare isolation of V. parahaemolyticus from seafood samples.