Abstract: AIM: To confirm the hypothesis that the cytotoxicity of tanshinone IIA (TSA) is dependent on NAD(P)H quinone oxidoreductase (NQO1)-mediated biotransformation. METHOD: MTT assay in a series of cell lines with diverse NQO1 enzyme activity in the presence or absence of the typical NQO1 inhibitor dicoumarol (DIC) was applied to test the NQO1-dependent cytotoxicity of TSA. Then the NQO1-dependent biotransformation of TSA in HepG2 cells was investigated with or without DIC. RESULTS: TSA's cytotoxicity is positively correlated with NQO1 enzyme activity in the cell lines. DIC could reverse the cytotoxicity of TSA in HepG2 cells with the highest NQO1 enzyme. DIC could also inhibit the biotransformation of TSA in HepG2 cells. CONCLUSION: By inhibiting NQO1, DIC could inhibit the biotransformation of TSA into a catechol intermediate in HepG2 cells, resulting in disturbance of the redox cycle of TSA, thereby reducing the cytotoxicity of TSA. In summary, NQO1-mediated biotransformation determines the cytotoxicity of Tanshinone IIA.