We investigated the impacts of exopolysaccharides isolated from Bacillus velezensis SN-1 (referred to as rEPS-2) on human hepatocellular carcinoma HepG2 cells, including their influence on apoptosis. HepG2 cells were treated with rEPS-2 for 48 hours, and their morphological characteristics were observed using an inverted microscope and fluorescence microscopy. An MTT assay was employed to examine the effects of rEPS-2 treatment for 24 and 48 hours on HepG2 cell survival and proliferation. The impact of treatment on cell cycle distribution was analyzed by flow cytometry. The inhibitory effects of rEPS-2 on HepG2 cells were time and dose dependent. At an rEPS-2 concentration of 2 mg•mL-1, inhibition of cell proliferation reached 72.35%. Flow cytometry analysis using Annexin V/PI double staining indicated dose dependent induction of G1/S phase arrest, with an increased percentage of cells in G0/G1 phase. rEPS-2 also increased the activity of apoptosis-associated enzymes Caspase-9 and Caspase-3. After 48 hours of treatment with rEPS-2, Caspase-3 and Caspase-9 activities increased to 140% and 162%, respectively. In conclusion, rEPS-2 displays significant in vitro antiproliferative efficacy against HepG2 cells. These findings provide a reference and supportive data for rEPS-2 development and application.