Based on microarray data, we have previously shown a significant down‐regulation of miR‐29 in hepatocellular carcinoma (HCC) tissues. To date, the role of miR‐29 deregulation in hepatocarcinogenesis and the signaling pathways by which miR‐29 exerts its function and modulates the malignant phenotypes of HCC cells remain largely unknown. In this study, we confirmed that reduced expression of miR‐29 was a frequent event in HCC tissues using both Northern blot and real‐time quantitative reverse‐transcription polymerase chain reaction. More interestingly, we found that miR‐29 down‐regulation was significantly associated with worse disease‐free survival of HCC patients. Both gain‐ and loss‐of‐function studies revealed that miR‐29 could sensitize HCC cells to apoptosis that was triggered by either serum starvation and hypoxia or chemotherapeutic drugs, which mimicked the tumor growth environment in vivo and the clinical treatment. Moreover, introduction of miR‐29 dramatically repressed the ability of HCC cells to form tumor in nude mice. Subsequent investigation characterized two antiapoptotic molecules, Bcl‐2 and Mcl‐1, as direct targets of miR‐29. Furthermore, silencing of Bcl‐2 and Mcl‐1 phenocopied the proapoptotic effect of miR‐29, whereas overexpression of these proteins attenuated the effect of miR‐29. In addition, enhanced expression of miR‐29 resulted in the loss of mitochondrial potential and the release of cytochrome c to cytoplasm, suggesting that miR‐29 may promote apoptosis through a mitochondrial pathway that involves Mcl‐1 and Bcl‐2. Conclusion: Our data highlight an important role of miR‐29 in the regulation of apoptosis and in the molecular etiology of HCC, and implicate the potential application of miR‐29 in prognosis prediction and in cancer therapy. (HEPATOLOGY 2010.)