Abstract One obstacle of cancer therapy is the development of cancer resistance to chemotherapy. The molecular mechanisms by which the resistance is developed remain to be fully understood. Apoptosis repressor with caspase recruitment domain (ARC) is an endogenous antiapoptotic protein. Here, we report that ARC contributes to chemotherapy resistance by abolishing mitochondrial fission mediated by dynamin-related protein-1 (Drp1). Our results show that both HeLa and human gastric cancer (SGC-7901) cells have a high expression level of ARC. Doxorubicin at a low dose can slightly induce apoptosis in HeLa and SGC-7901 cells. In contrast, knockdown of ARC by its RNA interference enables the same low dose of doxorubicin to significantly induce apoptosis in HeLa and SGC-7901 cells. These data indicate that ARC is responsible for the cell resistance to doxorubicin treatment. Mitochondrial fission has recently been shown to be involved in triggering apoptosis. In exploring the molecular mechanism by which ARC participates in antagonizing doxorubicin-induced apoptosis, we observed that doxorubicin is able to induce mitochondrial fission that can be inhibited by ARC. Our results further show that Drp1 accumulates in mitochondria and mediates the signal of doxorubicin to induce mitochondrial fission. ARC is able to prevent Drp1 accumulations in mitochondria. Finally, we identified that PUMA is required for Drp1 accumulations in mitochondria. ARC inhibits Drp1 accumulations in mitochondria by directly binding to PUMA. Taken together, our results reveal a chemotherapy-resistant model in which ARC inhibits PUMA-mediated Drp1 accumulations in mitochondria and the consequent mitochondrial fission. [Cancer Res 2009;69(2):492–500]