The multikinase inhibitor sorafenib (SRF) is approved for the treatment of renal and hepatic carcinomas and is also undergoing evaluation in therapeutic combinations with other anticancer agents. SRF is generally well tolerated but produces severe toxicities in a significant proportion of patients by mechanisms that are largely unknown. It has been shown that cytochrome P450 (CYP) 3A4 has a major role in SRF biotransformation to the pharmacologically active N-oxide (SRF-Nox) and two other metabolites. In this study, we prepared the major metabolites of SRF and evaluated their further biotransformation by CYPs in relation to their capacity to produce cellular toxicity. CYP3A4 was also found to be the principal enzyme that mediated the secondary oxidation of SRF metabolites. However, the reduction of SRF-Nox to SRF was also found to be a significant reaction mediated by several CYPs, especially CYPs 2B6 and 1A1. In human liver-derived HepG2 cells, SRF effectively decreased ATP production to an extent greater than that of its metabolites. SRF also markedly altered the cell cycle distribution in HepG2 cells by decreasing the proportion in G0/G1 phase and increasing that in S and G2/M phases. In comparison, SRF metabolites minimally affected HepG2 cell cycle progression. These findings suggest that SRF, but not its metabolites, prevents cells from entering the cell cycle and also inhibits cycling cells from completing mitosis. Reduction of the major metabolite SRF-Nox back to SRF may mediate decreased cellular viability and contribute to adverse reactions in some individuals.