Preserved egg is a traditional Chinese food, and its hydrolysates possess anti-oxidant, anti-inflammatory and anti-cancer effects, which has promising medical prospects. However, the anti-cancer efficacy and mechanism of preserved egg are still unclear. This study investigated the influence of preserved egg simulated gastrointestinal digests (PESD) on cell proliferation and cell cycle of human hepatocellular carcinoma HepG2 cells. After in vitro digestion treatment of preserved eggs by simulated gastrointestinal tract, a cell experiment was conducted. The CCK-8 method was employed to determine the effect of PESD on cell proliferation. Propidium iodide (PI) staining was used to detect the effect of PESD on HepG2 cells cycle by flow cytometry, and the expression of cell cycle regulatory proteins was determined by western blot. The results showed that the HepG2 cells proliferation was inhibited by PESD in a dose-dependent manner, and the IC₅₀ was 4.17mg/mL. The PESD significantly increased the proportion of cells in S phase (P<0.05), and blocked the cell cycle in S phase. The protein expression levels of cyclin A, cyclin E, ATM, chk2 and CDC25A increased (P<0.05), while the protein expression of CDK2 decreased (P<0.05) after 24h treatment with 4mg/mL PESD. Therefore, it was concluded that PESD could significantly inhibit the proliferation of HepG2 cells in a dose-dependent manner. It could up-regulate the expression of cyclin A and cyclin E protein and down-regulate CDK2 protein expression through the ATM-chk2-CDC25A signaling pathway to block the HepG2 cells cycle in S phase, affect the HepG2 cells cycle progress, and inhibit the proliferation of cancer cells. The results of the study might provide a theoretical basis for the elucidation of the anti-cancer mechanism of preserved eggs.