Mackerel (Pneumatophorus japonicus) protein was hydrolyzed by five proteases and the mackerel protein hydrolysate (MPH) treated by neutrase exhibited the most excellent antioxidant activity. The MPH was fractioned using ultrafiltration membranes and five fractions were obtained, furthermore, the MPHs with molecular weight below 2.5 kDa (MPH-III, MPH-IV and MPH-V) exhibited higher antioxidant activities. Finally, the MPHs was purified by SP Sephadex C-25 and Sephadex G-25, and the peptide I (MW=1664 Da) displayed the highest DPPH radical scavenging activity. In addition, the mechanism of MPHs for anti-fatigue were investigated in mice, the result showed that the MPHs groups could significantly prolong the exhaustion swimming time; the liver glycogen level was markedly increased and the BUN, LA and MDA levels were decreased in MPHs group compared to the normal group, respectively. In addition, the MPHs improved enzymatic antioxidant system by increasing the activities of SOD and GSH-Px. The results suggest that MPHs can be used as a potential source of antioxidant peptides.