The bioactive lipid sphingosine‐1‐phosphate (S1P) regulates smooth muscle ( SM) contractility predominantly via three G protein‐coupled receptors. The S1P1 receptor is associated with nitric oxide ( NO)‐mediated SM relaxation, while S1P2 & S1P3 receptors are linked to SM contraction via activation of the Rho‐kinase pathway. This study is to determine testosterone (T) modulating the expression and functional activity of S1P receptors in corpus cavernosum ( CC). Adult male Sprague‐Dawley rats were randomly divided into three groups: sham‐operated controls, surgical castration and T supplemented group. Serum S1P levels were detected by high‐performance liquid chromatography. The expression of S1P1‐3 receptors and sphingosine kinases was detected by real‐time RT‐ PCR. In vitro organ bath contractility and in vivo intracavernous pressure ( ICP) measurement were also performed. T deprivation significantly decreased ICP rise. Meanwhile, surgical castration induced a significant increase in serum S1P level and the expression of S1P2‐3 receptors by twofold ( P < 0.05) but a decrease in the expression of S1P1 receptor. Castration also augmented exogenous phenylephrine ( PE), S1P, S1P1,3 receptor agonist FTY720‐P contractility and S1P2‐specific antagonist JTE013 relaxation effect. T supplemented could restore the aforementioned changes. We provide novel data that castration increased serum S1P concentration and up‐regulated the expression of S1P2‐3 receptors in CC. Consistently, agonizing S1P receptors induced CCSM contraction and antagonizing mediated relaxation were augmented. This provides the first clear evidence that S1P system dysregulation may contribute to hypogonadism‐related erectile dysfunction ( ED), and S1P receptors may be expected as a potential target for treating ED.