Hypersensitivity of Brca1-deficient cells to interstrand crosslinking (ICL) agents such as cisplatin and mitomycin C (MMC) implicates an important role for Brca1 in cellular response to the ICL DNA damage repair. However, the detailed mechanism of how Brca1 is involved in the ICL response remains unclear. In this study, we analysed the cellular response to MMC treatment using isogenic mouse embryonic fibroblasts (MEFs) including wild type, p53−/− and p53−/−Brca1−/−. Marked hypersensitivity of p53−/−Brca1−/− MEFs to MMC was found, and the reconstitution of Brca1 expression in these cells restored resistance to MMC. Upon MMC treatment, wild-type MEF was temporarily arrested at G2/M phase but subsequently resumed a normal cell cycle progression. In contrast, Brca1-deficient MEF exhibited a marked time-dependent accumulation of cells arrested at S phase and a prolonged increase in the G2/M population, followed by extensive cell deaths. Importantly, DNA damage-induced Rad51 foci were not formed in these cells, suggesting a defect in homologous recombination. Such defects are fully rescued by reconstitution of Brca1 expression in Brca1-deficient MEF, suggesting that Brca1 directly plays an essential role in ICL repair, which depends on homologous recombination during S phase.