Fueled by the rapid growth of the global companion animal industry, the development of nutritional additives for improving canine intestinal health has emerged as a prominent research area in pet nutrition. Excessive reactive oxygen species accumulation triggers severe oxidative stress, inducing intestinal inflammation, abnormal epithelial senescence, and tight junction destruction to damage intestinal mucosal integrity. Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) have proven antioxidative and anti-inflammatory potency, yet divergent functions of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on canine ileal epithelial cells (cIECs) remain unclear. Here, an H₂O₂-stimulated oxidative injury model was built to compare their protective capacity. Under all equivalent gradient concentrations ranging from 2.5 to 10 μM in this experiment, Equal-dose DHA failed to alleviate cell damage, while EPA effectively rescued impaired cIEC viability. Biochemical tests showed EPA lowered malondialdehyde and boosted key antioxidant enzyme activities. Transcriptomics confirmed EPA suppressed overactivated TNF-α/IFN-γ inflammatory and senescence pathways. Western blot verified EPA downregulated p-Chk1, p53, p57, and p73 and restored telomere-protective and tight junction proteins. In summary, EPA protects cIECs via antioxidation, anti-inflammation and anti-senescence, supporting its development as a promising functional feed additive for canine intestinal care.