Abnormal high activation of survivin is involved in carcinogenesis of various types of cancer. Survivin has been shown to promote cell proliferation in human hepatocellular carcinoma (HCC). Survivin-targeting approaches have become a promising strategy for treating HCC. Here, we used a reporter system to screen effective survivin siRNA sequences. The effect of vector-based survivin short hairpin RNA (shRNA) on the malignant phenotype of HCC cells in vitro and in vivo was determined, and an adenovirus-mediated shRNA expression vector was developed to decrease survivin expression of the established HCC tumor in nude mice. In vitro study showed that stable survivin knockdown inhibited cancer cell proliferation, enhanced apoptotic susceptibility, arrested cell cycle in the G1 phase and resulted in apparent mitotic catastrophe. Moreover, cells stably expressing survivin shRNA showed decreased tumorigenicity in nude mice. An additional in vivo study showed that intratumoral injection of adenovirus-delivered survivin shRNA suppressed tumor growth by spontaneous apoptosis of cancer cells and significantly prolonged animal survival. In conclusion, we proved the therapeutic potential of survivin shRNA for the treatment of HCC. And our results indicated that adenovirus-delivered shRNA may serve as a novel therapeutic for HCC.