RhoGTPase-activating proteins (RhoGAPs) regulate RhoGTPases in cells, but whether individual reactive oxygen species (ROS) regulate RhoGAPs is unknown. Our previous published papers have shown that deleted in liver cancer 1 (DLC1) inhibits cancer cell migration by its RhoGAP activity. The present study was designed to explore the role of H2O2 in regulation of DLC1. We treated cells with H2O2 for 24h and phenotypic changes were analyzed by MTT, RT-PCR, Western blotting, immunofluorescence staining and wound healing assays. H2O2 downregulated cyclin D1 and cyclin E to inhibit proliferation, and upregulated BAX to induce apoptosis in MCF-7 cells. Compared with non-tumorigenic cells, H2O2 increased expression of DLC1 and reduced activity of RhoA in cancer cells. Stress fiber production and migration were also suppressed by H2O2 in MDA-MB-231 cells. Our study suggests that H2O2 inhibits proliferation through modulation of cell cycle and apoptosis-related genes, and inhibits migration by decreasing stress fibers via DLC1/RhoA signaling.