Oncoprotein p28GANK knockdown by RNA interference (RNAi) can induce hepatoma cells apoptosis. However, the mechanisms have not been well defined yet. In the present study the p28GANK knockdown-induced apoptosis in HepG2 cells was prevented by caspase-9 inhibitor (Z-LEHD-FMK). During the knockdown of p28GANK, mitochondrial translocation of Bax, loss of mitochondrial transmembrane potential (DeltaPsim) and release of cytochrome c were observed. In this study, the activation of p38 was found to be critical for the p28GANK knockdown-induced apoptosis, as suggested by the finding that pharmacological inhibition of p38 with SB203580 suppressed the redistribution of Bax, the loss of DeltaPsim and the apoptosis. Moreover, generation of reactive oxygen species (ROS) contributed to the cell death because N-acetyl-L-cystenine (NAC), a ROS scavenger, suppressed the phosphorylation of p38 and the apoptosis. Our studies established the signaling pathway of p28GANK knockdown-induced apoptosis in HepG2 cells, namely, mitochondrial dysfunction mediated by p38 downstream of intracellular ROS generation.