Objective To investigate the expression of SETDB1 in hepatocellular carcinoma (HCC) and its role in tumor growth. Methods The expression of SETDB1 in HCC was detected by immunohistochemical staining. SETDB1 specific shRNA-lentivirus was used to knock down the expression of SETDB1 in MHCC97H cells. Clone formation assay and flow cytometry were used to detect cell proliferation and apoptosis. A subcutaneous xenotransplanted tumor model was used to measure the effect of SETDB1 on tumor growth in vivo . Western blot was used to detect the expression of H3K9me3. Results The expression of SETDB1 in the HCC tissues was higher than that in the normal liver tissues (P < 0.05). The tumor volume was bigger in the patients with high SETDB1 expression (P < 0.05). Down-regulation of SETDB1 suppressed proliferation and enhanced apoptosis in MHCC97H cells (P < 0.05). Knockdown of SETDB1 still inhibited in vivo tumor growth in nude mice (P < 0.05). Moreover, the expression of H3K9me3 in MHCC97H cells was impaired due to the depletion of SETDB1 (P < 0.05), but the p53 expression was up-regulated (P < 0.05). Conclusions The expression of SETDB1 increases in HCC tissues. Downregulation of SETDB1 could inhibit HCC growth in vitro and in vivo through inhibiting H3K9 trimethylation.