Hepatocellular carcinoma (HCC) is a malignant tumor with high morbidity and mortality rates. E2F2 is an independent predictor of poor prognosis in HCC; however, The mechanism by which E2F2 promotes the progression of HCC remains unclear. The Shentao Ruangan (STR) formula exhibits antitumor efficacy against HCC; however, the underlying antitumor mechanisms remain unknown.To explore the regulatory effect of E2F2 on the p53 signaling pathway and reveal the role and mechanism of STR in promoting cell apoptosis via the E2F2-p53 signaling pathway in HCC.E2F2 overexpression or silencing by lentivirus in HepG2 cells were used to explore their influence on apoptosis and the p53 pathway. An H22 tumor-bearing mice model was used to determine the therapeutic efficacy of STR and its effects on the E2F2-p53 pathway. STR-mediated serum (STR-MS) was prepared, and its chemical constituents were identified using mass spectrometry. The effects of STR-MS on viability and apoptosis of HepG2 cells and the E2F2-p53 pathway were investigated and validated using rescue experiments.E2F2 overexpression significantly inhibited apoptosis and the p53 pathway in HepG2 cells, whereas E2F2-silenced HepG2 cells showed the reverse. This increased apoptosis was rescued by the addition of a p53 inhibitor (PFT-α) to E2F2-silenced HepG2 cells. In vivo, high doses of STR could remarkably inhibit the growth of xenografts, promote the apoptosis of hepatoma cells, downregulate E2F2, and activate the p53-dependent mitochondrial apoptotic pathway with good safety. In vitro, STR-MS exhibited similar effectiveness, and the best effect was achieved at 30% STR-MS concentration for 48 h. When 30% STR-MS was added to E2F2-overexpressing cells, the increased apoptosis and expression of key proteins in the p53-dependent mitochondrial apoptosis pathway were significantly rescued.Our findings demonstrate, for the first time, that E2F2 inhibits hepatoma cell apoptosis in a p53-dependent manner and that STR may promote apoptosis by regulating the E2F2-p53 pathway in HCC.Copyright © 2022. Published by Elsevier GmbH.