Hepatitis B x-interacting protein (HBXIP), a co-factor of survivin, was originally identified by its binding with the C-terminus of the hepatitis B virus x protein (HBx). We have recently shown that HBXIP promotes the growth of both normal liver cells and hepatoma cells in vitro, but the molecular mechanisms of this have not been documented. In this study, we investigated the potential effects of HBXIP on the proliferation of HepG2 cells and the intracellular signaling pathway mediating these changes. Over-expression of the HBXIP gene promoted the proliferation of HepG2 cells, as shown by the MTT assay. We also showed that HBXIP induced cellular accumulation in the S phase concomitantly with up-regulation of cyclinD(1) and down-regulation of p21 and p53 levels. Moreover, HBXIP over-expression cells showed activation of the PI3K/Akt pathway; this activation was accompanied by an increase in phosphorylation of glycogen synthase kinase 3β. LY294002, a specific inhibitor of PI3K, blocked HBXIP-stimulated Akt phosphorylation and suppressed the cell cycle promotion induced by HBXIP in HepG2 cells. The increase in cyclinD(1) protein levels induced by HBXIP was inhibited when cells were incubated with LY294002. In conclusion, our data suggest that the proliferation of HepG2 cells promoted by HBXIP is associated with activation of the PI3K/Akt signaling pathway.