[Objective] To elucidate differences of biological characteristics and cytopathogenicities between T3SS1 and T3SS2 in Vibrio parahaemolyticus. [Methods] With the main structural protein genes vcrD1 and vcrD2 as the research object, we used the homologous recombination technique to construct a series of deletion mutants ΔvcrD1, ΔvcrD2, ΔvcrD1-vcrD2, and complementary strains CΔvcrD1, CΔvcrD2 of V. parahaemolyticus SH112 strain, then analyzed the differences of growth, biofilm, motility, cytotoxicity to RAW264.7 macrophages and Caco-2 cells, and transcriptional regulation of pro-inflammatory cytokine IL-1β, IL-6 in RAW264.7 macrophages between mutant strain and wild strain. [Results] Compared to the wild strain, the mutant strains had no difference on growth rate, however, the vcrD1 mutant obviously weakened biofilm formation, motility, cytotoxicity of V. parahaemolyticus; the vcrD2 mutant positively upregulated the transcriptional levels of pro-inflammatory cytokines, and significantly attenuated cytotoxicity to cells. The ΔvcrD1-vcrD2 double deletion mutant showed weaker biofilm, motility, cell toxicity than vcrD1 mutant strain, and as similar to the vcrD1 mutant, in the mRNA levels of inflammatory cytokines when compared with the wild strain. [Conclusion] Contributions of two T3SSs to biological characteristics and cytopathogenicities of V. parahaemolyticus are different. The T3SS1 was mainly involved in biofilm formation and motility of V. parahaemolyticus, but also has a significant cytotoxic effect; T3SS2 did not affect bacterial biofilm formation and motility, but played a negative regulation on cell inflammatory reaction, potentially contributing to bacterial immune evasion in host. T3SS1 contributed to bacterial survival in the environment; T3SS2 may help V. parahaemolyticus to evade the host immune response. Moreover, T3SS1 and T3SS2 may play related functions on biological characteristics and cytopathogenicities of V. parahaemolyticus, but the specific mechanism still needs further study.