Omi/HtrA2 promotes cell apoptosis in human cancer cells. Early studies showed that primary hepatocellular carcinoma requires Omi/HtrA2 expression for cell apoptosis. Additionally, the Omi/HtrA2 pro-apoptotic marker demonstrated a difference in some cell types. However, how the Omi/HtrA2 pro-apoptotic marker reacts during the process of hepatocellular carcinoma cell apoptosis remains to be determined. Thus, we investigated the role and possible mechanism of Omi/HtrA2 on hepatocellular carcinoma cell apoptosis using various hepatocellular carcinoma cell lines. The results were analyzed using RT-qPCR and western blot analysis. In the present study, we found that Omi/HtrA2 was overexpressed in hepatocellular carcinoma cell lines and induced hepatocellular carcinoma cell apoptosis. Additiionally, the only manner in which Omi/HtrA2 participated in cell death in PLC cells may be dependent on IAP-binding. Omit HtrA2-inducing HepG2 cell apoptosis may mainly depend on its serine protease activity while both IAP-binding and its serine protease activity participated in Hep3B cell apoptosis. This result suggested that Omi/HtrA2 pro-apoptotic marker differs in various hepatocellular carcinoma cell lines. PLC cells were also devoid of the expression of ped/pea-15 as the substrate of Omi/HtrA2 serine protease while ped/pea-15 was overexpressed in HepG2 and Hep3B cells and ped/pea-15 expression was higher in HepG2 cells than that in Hep3B cells. These results showed that Omi/HtrA2 overexpression promotes hepatocellular carcinoma cell apoptosis and the ped/pea-15 expression level causes this difference of the Omit HtrA2 pro-apoptotic marker in the various hepatocellular carcinoma cell lines.