Objective To explore the role and mechanism of inhibitor of differentiation 1 (Id1) on replication of the hepatitis B virus, and proliferation and apoptosis of hepatoma cells. Methods The Id1 gene in the HepG2. 2. 15 hepatoma cell line was silenced by RNA interference (siRNA-Id1 group). An unrelated sequence was designed and used for the siRNA-Control group. The expression of p53 in siRNA-Id1 cells was inhibited by pifithrin-α ( siRNA-Id1+Pifithrin-α group) and HepG2. 2. 15 hepatoma cells were used as the control group. The replication of hepatitis B virus, cell proliferation, and apoptosis were detected by qRT-PCR, western blot, ELISA, MTT, and flow cytometry, respectively. Results The mRNA and protein expressions of Id1 in the siRNA-Id1 group were lower than those in the siRNA-Control group (P<0. 001). Compared with the siRNA-control group, the mRNA and protein expressions of HBx in the siRNA-Id1 group were decreased (P<0. 001), the contents of HBsAg and HBeAg in the supernatant were decreased (P<0.001), the proliferation rate of cells was decreased (P<0. 05), the proportions of early apoptosis cells and late apoptosis cells were increased (P<0. 001), the protein expressions of Bax and cleaved caspase-3 in cells were increased (P<0. 001), and the protein expression of BcL-2 was decreased (P<0. 05). Pifithrin-α partly inhibited these biological effects induced by Id1 gene silencing. Conclusions The RNA interference of Id1 reduced the replication of hepatitis B virus, inhibited the proliferation of hepatocellular carcinoma cells, and induced cell apoptosis. The mechanism may be related to the overexpression of p53.