Abstract: To provide the profiles of metabolism of mitomycin C (MMC) by human liver microsomes in vitro, MMC was incubated with human liver microsomes, then the supernatant component was isolated and detected by HPLC. Types of metabolic enzymes were estimated by the effect of NADPH or dicumarol (DIC) on metabolism of MMC. Standard, reaction, background control (microsomes was inactivated), negative control (no NADPH), and inhibitor group (adding DIC) were assigned, the results were analyzed by Graphpad Prism 4.0 software. Reaction group compared with background control and negative control groups, 3 NADPH-dependent absorption peaks were additionally isolated by HPLC after MMC were incubated with human liver microsomes. Their retention times were 10.0, 14.0, 14.8 min (named as M1, M2, M3), respectively. Their formation was kept as Sigmoidal dose-response and their Km were 0.52 (95% CI, 0.40-0.67) mmol·L^-1, 0.81 (95% CI, 0.59-1.10) mmol·L^-1, 0.54 (95% CI, 0.41-0.71) mmol·L^-1, respectively. The data indicated that the three absorption peaks isolated by HPLC were metabolites of MMC. DIC can inhibit formation of M2, it’s dose-effect fitted to Sigmoidal curve and it’s IC₅₀ was 59.68 (95% CI, 40.66-87.61) μmol·L^-1, which indicated DT-diaphorase could take part in the formation of M2. MMC can be metabolized by human liver microsomes in vitro, and at least three metabolites of MMC could be isolated by HPLC in the experiment, further study showed DT-diaphorase participated in the formation of M2.