High temperature can reduce testes function, leading to decreased testosterone secretion. Dietary l-arginine (l-Arg) supplementation improves the semen quality and libido of boars. The present study investigated whether l-Arg could enhance the production of testosterone in mice exposed to high ambient temperature. Twenty-four 6-week-old male ICR mice were randomly divided into three groups: a control group, a heat-treated (HT) group and a group subjected to heat treatment plus 2mg kg-1 l-Arg (HT+Arg). l-Arg was administered to mice by oral gavage for 18 consecutive days, after which the HT and HT+Arg groups were placed into an incubator at 40°C for 30min every day for 5 days. Serum testosterone and LH concentrations were significantly increased in the HT+Arg compared with HT group, as was catalase, total superoxide dismutase and glutathione peroxidase activity and the expression of steroidogenesis-related genes steroidogenic acute regulatory protein (Star), steroidogenic factor-1 (Sf1), 17β-hydroxysteroid dehydrogenase 3 (Hsd17b3) and 17α-hydroxylase/17,20-lyase (Cyp17a1) in the testes. These results demonstrate that l-Arg can alleviate testosterone reductions in heat-treated mice by upregulating LH secretion, enhancing the antioxidant system and increasing the expression of testosterone synthesis-related genes.