The effect of photobiomodulation on delayed onset muscle soreness remains unknown. This study represents the first investigation of this treatment using an animal model. Seventy-two Sprague-Dawley rats were randomly divided into five groups: sedentary control group, exercise control group and three exercise-plus-laser groups. Downhill running was used to induce muscle injury in the gastrocnemius muscle. He-Ne laser irradiations were administered to the injured muscles immediately and at 18 and 42 h after exercise in the three exercise-plus-laser groups at 12, 28, and 43 J/cm2, respectively. Histological examination and serum creatine kinase (CK), muscle superoxide dismutase (SOD) and malondialdehyde (MDA) analyses were done at 24 and 48 h after exercise. The exercise control group exhibited a marked inflammation in the gastrocnemius muscle and significant elevations in serum CK activity and muscle MDA level after downhill running. He-Ne laser irradiation at 43 J/cm2 inhibited muscle inflammation, significantly enhanced muscle SOD activity and significantly reduced serum CK activity and muscle MDA level at both 24 and 48 h after exercise, whereas the irradiation at 12 or 28 J/cm2 slightly inhibited muscle inflammation and significantly reduced serum CK activity at 48 h after exercise only (P<0.05). Low-level He-Ne laser therapy could exert therapeutic effects on eccentric exercise-induced rat muscle injury through enhancing muscle anti-oxidative capacity and reducing the inflammatory reaction. The photobiomodulation was dose-dependent, and the 43 J/cm2 dose was the most efficient among the doses used.