Toll-like receptor (TLR) 2 signaling is regarded as one of the mechanisms of chronic inflammation, but it can also mediate tumor cell immune escape and tumor progression. However, the role of TLR2 in the progression of human hepatocellular carcinoma (HCC) remains unclear. The objective of the study was to examine the effect of TLR2 on the bioactivity of HCC cell lines, HepG2 and BEL-7402, and the relationship between high mobility group box1 (HMGB1) and TLR2. The expression of TLR2 and nuclear factor-kappaB/P65 (NF-κB/P65) in HepG2 and BEL-7402 was assayed by Western blot. Cells were transfected with specific small interfering RNAs of TLR2 (TLR2-siRNAs), then TLR2-siRNA-transfected cells were treated with recombinant HMGB1 (rHMGB1). Apoptosis was determined by flow cytometry. Results showed that TLR2 was expressed in HepG2 and BEL-7402 cells. The ability of proliferation, invasion, and migration in siRNA group was lower than that in blank group, and the apoptosis ratio was higher than that in blank group, respectively. NF-κB/P65 expression was declined in contrast with blank group. Downregulation of TLR2 by siRNA resulted in a significant inhibition of proliferation, invasion, migration, and NF-κB/P65 expression, and elevated apoptotic ratio. Conversely, rHMGB1 promoted proliferation, invasion, and migration, induced NF-κB/P65 expression, and inhibited cells apoptosis. Furthermore, downregulation of TLR2 weakened the role of rHMGB1. This study suggests TLR2 and HMGB1 are important targets for therapeutic intervention of HCC.