The aim of the study was to investigate how testosterone regulating endothelial function in the corpus cavernosum of rats. A total of 32 male Sprague-Dawley (SD) rats, 10-weeks-old, were divided randomly into four groups: normal control group (Control); castration group (Castration); the other 16 rats were castrated followed by testosterone supplementation (orally) every day for 8 weeks: castration +10 mg/kg (lower dose) testosterone group (Castration +LT) and castration +20 mg/kg (high dose) testosterone group (Castration +HT). The data showed that androgen deficiency in the Castration group could induce oxidative stress to attenuate endothelial function, manifested by the impairment of endothelial intercellular junction and endothelial content. This was in parallel with a significant decrease in Akt, Akt target and FOXO3a phosphorylation. Testosterone supplementation in the Castration +LT and Castration +HT groups could greatly preserve testosterone serum levels, endothelial function and erectile function through activation of sphingosine-1-phosphate receptor 1 (S1P1)/Akt/FOXO3a pathway. Together, this study suggested that S1P1/Akt/FOXO3a pathway was involved in endothelial dysfunction in the context of androgen deficiency and oxidative stress, which might further explain the mechanism of androgen deficiency inducing ED.