To investigate the effects of procyanidin B2 (PB2) on the proliferation of hepatocellular carcinoma (HCC) cells and its potential molecular mechanism, this study employed cell counting kit-8 (CCK-8), calcein acetoxymethyl ester /propidium iodide (Calcein-AM/PI) staining, and colony formation assay to assess the effects of PB2 on the proliferation of Hep3B and Huh7 cells. Flow cytometry was used to observe changes in the cell cycle, immunofluorescence was utilized to detect the expression level of phosphorylated H2A histone family member X (γ-H2AX), and Western blot (WB) was performed to evaluate the expression levels of cell cycle and deoxyribonucleic acid (DNA) damage-related proteins. The results showed that PB2 significantly inhibited the proliferation of HCC cells and the expression level of proliferating cell nuclear antigen (PCNA) was decreased. After 24 h of PB2 treatment, the proportion of cells in the S-phase significantly increased without inducing cell death. Meanwhile, the expression levels of the cell cycle-related proteins cyclin-dependent kinase 2 (CDK2), Cyclin A, and Cyclin D1 significantly decreased, suggesting cell cycle arrest; the expression level of γ-H2AX protein was significantly upregulated, indicating significant DNA damage. Notably, the DNA protector methylphenidate (MPA) could partially reverse these phenomena, potentially indicating that PB2 inhibits the proliferation of HCC cells by inducing DNA damage and cell cycle arrest. This study provides an experimental basis for the application of PB2 in HCC treatment research.