The human transmembrane 4 superfamily member 4 or intestinal and liver tetraspan membrane protein (TM4SF4/il-TMP) was originally cloned as an intestinal and liver tetraspan membrane protein and mediates density-dependent cell proliferation. The rat homolog of TM4SF4 was found to be up-regulated in regenerating liver after two-thirds hepatectomy and overexpression of TM4SF4 could enhance liver injury induced by CCl(4). However, the expression and significance of TM4SF4/il-TMP in liver cancer remain unknown. Here, we report that TM4SF4/il-TMP is frequently and significantly overexpressed in hepatocellular carcinoma (HCC). Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis showed that TM4SF4/il-TMP mRNA and protein levels were up-regulated in ∼80% of HCC tissues. Immunohistochemical analysis of a 75 paired HCC tissue microarray revealed that TM4SF4/il-TMP was significantly overexpressed in HCC tissues (P< 0.001), and high immunointensity of TM4SF4/il-TMP tended to be in well-to-moderately differentiated HCC compared with poorly differentiated tumors. Functional studies showed that overexpression of TM4SF4/il-TMP in QGY-7701 and BEL-7404 HCC cell lines through stable transfection of TM4SF4 expression plasmid significantly promoted both cell growth and colony formation of HCC cells. Reduction of TM4SF4/il-TMP expression in QGY-7701 and BEL-7404 cells by stably transfecting TM4SF4 antisense plasmid caused great inhibition of cell proliferation. Our findings suggest that TM4SF4/il-TMP has the potential to be biomarker in HCC and plays a crucial role in promotion of cancer cell proliferation.