This study aims to investigate the effect of ω-3 fatty acid supplementation on the recovery capacity of mice undergoing high-intensity interval training (HIIT). A total of 48 male C57BL/6 mice, aged eight weeks, were randomly divided into four groups: control group (Con), model group (Mod), low dose ω-3 fatty acid group (L-ω-3, 100 mg/kg/d), and high dose ω-3 fatty acid group (H-ω-3, 300 mg/kg/d). All groups except Con group underwent a 4-week HIIT regimen while receiving ω-3 fatty acid intervention during modeling. After the experiment, serum levels of inflammatory factors, oxidative stress indicators in muscle tissue, glycogen content, muscle injury markers, mitochondrial function-related indicators, and exhaustion swimming time were measured. Muscle tissues were subjected to pathological examination, electron microscopy, and Western blot analysis. The results showed that compared to the Mod group, the ω-3 fatty acid supplementation groups significantly reduced serum levels of tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), and interleukin 6 (IL-6) in serum, increased the activity of superoxide dismutase (SOD) and the content of glutathione (GSH) in muscle tissue, decreased the content of malondialdehyde (MDA), increased muscle glycogen content, reduced the activity of serum creatine kinase (CK) and lactate dehydrogenase (LDH), enhanced mitochondrial ATP synthase activity and cytochrome C oxidase activity, and prolonged exhaustion swimming time (P<0.05, P<0.01). Mechanistic studies indicated that ω-3 fatty acids significantly improved apoptosis in muscle tissue, lowering the expression levels of cleaved Caspase-3, cleaved Caspase-8, Fas, and Fas L proteins (P<0.05, P<0.01). The findings suggested that ω-3 fatty acid supplementation effectively inhibited the inflammatory response in HIIT mice, alleviated oxidative stress, promoted energy reserves, protected muscle tissue, and enhanced mitochondrial function, thereby improving exercise recovery capacity.