Abstract. DNA methylation is an important regulator of gene transcription, and its role in carcinogenesis has been a topic of considerable interest in previous years. The present study examined the influence of DNA methyltransferase 3b (DNMT3b) on cell proliferation, migration and invasion, and the methylation status of identified tumor suppressor genes in hepatoma SMMC-7721 and BEL-7402 cells. DNMT3b was silenced by small interfering RNA (siRNA) in human hepatocellular carcinoma cell lines. Transfection efficiency was verified using a fluorescent imaging system, reverse transcription polymerase chain reaction (RT-PCR) and western blotting. A cell proliferation assay was performed to evaluate cell viability. Cell cycle distribution and apoptosis were analyzed by flow cytometry. The migratory and invasive ability of cells was measured using a Transwell assay. Methylation-specific PCR (MSP) was performed to assess methylation in the promoter region of genes. The present data revealed that DNMT3b siRNA successfully inhibited expression of the DNMT3b gene in these two liver cancer cell lines and therefore inhibited the proliferation of the transfected cells, stimulated apoptosis in the cells, led to an accumulation of cells in the G 2 /M phase and decreased cell migration and invasion. It was also found that silencing DNMT3b expression results in hypomethylation of specific sets of gene promoters and increases the expression of distinct set of genes in HCC cell lines. The present study is therefore useful for assessing the specificity of emerging action based on the altered expression of associated regulatory genes, particularly in methylation-silenced genes.