Proapoptotic BH3-only protein Bim plays an important role in Bax/Bak-mediated cytochrome c release and apoptosis. Here we provide evidence for a novel prosurvival function of Bim in cancer cells. Bim was constitutively overexpressed in multiple prostate and breast cancer cells as well as in primary tumor cells. Quantitative real time PCR analysis showed that Bim was transcriptionally upregulated. We have identified eight endogenous E2F1-binding sites on the Bim promoter using in silico analysis. Luciferase assay demonstrated that Bim expression was E2F1-dependent as mutation of E2F1-binding sites on the Bim promoter inhibited luciferase activities. In support, E2F1 silencing led to the loss of Bim expression in cancer cells. Bim primarily localized to mitochondrial and cytoskeleton-associated fractions. Bim-silencing or microinjection of anti-Bim antibodies into the cell cytoplasm resulted in cell rounding, detachment, and subsequent apoptosis. We observed upregulation of prosurvival proteins Bcl-xL and Mcl-1, which sequester Bim in cancer cells. In addition, phosphorylated form of Bim was also elevated in cancer cells. These findings suggest that the constitutively overexpressed Bim may function as a prosurvival molecule in epithelial cancer cells, and phosphorylation and association with Bcl-xL/Mcl-1 block its proapoptotic functions.