The proliferation of small intestinal epithelial cells is fundamental to intestinal development and barrier function maintenance in infants and young children. To investigate the effects and mechanisms of ω-3 polyunsaturated fatty acids (docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA)), commonly used in infant formula, on this process, this study employed the rat intestinal epithelial cell line 6 (IEC-6) as a model. After DHA and EPA treatment at different combinations of concentration (1, 10, 50, 100 μmol/L) and duration 12, 24, 36, 48 h, cell viability was assessed using the cell counting kit-8 and 5-ethynyl-2’-deoxyuridine assays, and the mRNA and protein expression of key molecules involved in the Wnt/β-catenin signaling pathways were analyzed via real-time quantitative polymerase chain reaction and Western blot. The results showed that both DHA and EPA significantly promoted the proliferation of IEC-6 cells, with optimal concentrations of 100 and 10 μmol/L, respectively, and an optimal treatment duration of 24 h. Specifically, treatment with 100 μmol/L DHA and 10 μmol/L EPA for 24 h increased cell viability by nearly 4- and 3.5-fold compared to the control group (P < 0.001), respectively. Both DHA and EPA activated the Wnt/β-catenin signaling pathway, significantly upregulating the mRNA and protein expression of cell proliferation-related genes such as cyclin D1 and β-catenin (P < 0.05, P < 0.01, or P < 0.001). These findings indicate that DHA and EPA enhance intestinal epithelial cell proliferation by activating the Wnt signaling pathway.