Objective: Hesperidin, an active constituent of traditional Chinese medicine, Chenpi, exhibits anticancer properties across different cancers. This study aimed to clarify the efficacy of Hesperidin against tumors and its mechanisms of action in colon cancer. Method: We assessed the efficacy of Hesperidin on human colon cancer cells (HCT-116 and DLD-1) and normal colonic epithelial cells (NCM460). We quantified cell viability at various Hesperidin concentrations using the CCK8 assay in a series of experiments. We employed clone formation, EdU incorporation, Transwell, and wound healing assays to clarify Hesperidin efficacy on cancer cell proliferation, invasion, and migration. Western blot analyses revealed modulations in epithelial-mesenchymal transition-related proteins, SLC5A1, EGFR, and phosphorylated EGFR levels following Hesperidin exposure. Co-IP assays further validated the interaction between SLC5A1 and EGFR. Our findings were significantly restored following SLC5A1 overexpression in colon cancer cells, highlighting its pivotal role in Hesperidin-induced responses. Results: Hesperidin selectively impaired the viability of HCT-116 and DLD-1 colon cancer cells at specific concentrations while preserving normal NCM460 cells. This flavonoid dose-dependently reduced cancer cell proliferation, migration, and invasion. It significantly suppressed SLC5A1 and phosphorylated EGFR expression. We identified a direct SLC5A1-EGFR interaction essential for regulating EGFR activity in colon cancer. Overexpressing SLC5A1 significantly reduced the inhibitory effects of Hesperidin, highlighting its crucial role in this context. Conclusion: Hesperidin exerts its anticancer effects on colon cancer by inhibiting SLC5A1 expression and consequently downregulating EGFR phosphorylation.