High hepatitis B virus (HBV) load and chronic hepatitis B infection increase the risk of developing hepatocellular carcinoma (HCC), and is also associated with recurrence of HBV-related HCC. The aim of the present study was to investigate whether pseudolaric acid B (PAB), a diterpene acid isolated from the root and trunk bark of Pseudolarix kaempferi Gordon (Pinaceae), has an inhibitory role on the HBV secretion in HBV-related HCC. By detecting HBV surface antigen (HBsAg) by ELISA it was found that PAB inhibited HBV secretion in HepG2215 compared to control group, but did not decrease the intracellular HBV level, and the results were repeated in HepG2 cell transfect with HBV gene. Therefore, our results proved that PAB had the ability to inhibit HBV secretion. Moreover, it was shown that HepG2215 cells with HBV gene accumulated more in G0/G1 phase than HepG2 cells without HBV gene through detecting cell cycle distribution by flow cytometry, which indicated that HBV replication might favor the cell cycle environment of G0/G1 phase. However, HepG2 cells entered G2/M phase earlier than HepG2215 when PAB treatment induced G2/M arrest, therefore, HBV retarded the entry of G2/M to sustain the status of G0/G1 phase, while PAB finally changed the cell cycle environment favored by HBV virus. In addition, PAB also induced HepG2215 cell apoptosis, which would be helpful to kill the cells infected by HBV and help for devouring HBV by macrophage. Therefore, PAB inhibited HBV secretion through apoptosis and cell cycle arrest. The present findings contribute to a future potential chemotherapeutic drug in the treatment of HBV-related HCC.