The role of T-type Ca2+ channels in hepatocellular carcinoma cell proliferation was investigated in vitro. Eleven hepatocellular carcinoma cell lines and one immortalized liver cell line (LO2) were examined for the status of T-type Ca2+ channels with RT-PCR and voltage-clamp recordings. Except HBxF344, other cell lines tested had one, two or all three of alpha1-subunits (alpha1G, alpha1H and alpha1I) mRNA expression. Obvious T-type current was recorded in SNU449 cells, while others exhibited a minimal or no T-type current. SNU449, PLC/PRF5, Hep3B and LO2 cell lines were subjected to growth assay in the presence of Mibefradil, a T-type Ca2+ channel blocker, only the proliferation of SNU449 cell which had functional T-type Ca2+ channel was reduced by Mibefradil treatment. Furthermore, the persistent increase of phosphorylated ERK1/2 in SNU449 cells was found when treated with Mibefradil. A microarray assay also demonstrated some down-regulated genes were mainly associated with cell cycle and cell proliferation in Mibefradil treated SNU449 cells. In conclusion, this study showed that the functional T-type Ca2+ channels probably participate in modulating the proliferation of some hepatocellular carcinoma cells. The cell proliferation reduction of SNU449 with Mibefradil treatment is possibly associated with the persistent increase of phosphorylated ERK1/2.