The increasing intensity of exercise enhanced corticosterone and lactate production in both humans and rodents. Our previous studies also demonstrated that lactate could stimulate testosterone production in vivo and in vitro. However, the production of testosterone in response to combined corticosterone and lactate on Leydig cells, and underlying molecular mechanisms are remained unclear. This study investigated the changes in testosterone levels of Leydig cells upon exposure to lactate, corticosterone or combination of both, and revealed the detailed mechanisms. Leydig cells were isolated from rat testes, and treated with different concentrations of lactate (2.5–20 mM), cortiosterone (10−9–10−4 M) and lactate plus corticosterone. The production of testosterone were assayed by radioimmunoassay, and the key molecular proteins, including luteinizing hormone receptor (LHR), protein kinase A (PKA), steroidogenic acute regulatory protein (StAR), and cholesterol P450 side‐chain cleavage enzyme (P450scc) involved in testosterone production were performed by Western blot. Results showed that testosterone levels were significantly increased with lactate, while decresed with corticosterone and lactate plus corticosterone treatment. Protein expressions of LHR and P450scc were upregulated with lactate treatment. However, PKA and P450scc were downregulated by lactate plus corticosterone treatment. This downregulation was followed by decreased testoterone levels in Leydig cells. Furthermore, acetylated cAMP, which activates testosterone production was increased with lactate, but not altered by conrtiosterone. Our findings conclude that corticosterone may interfere with lactate, and restrict lactate‐stimulated testosterone production in Leydig cells. J. Cell. Physiol. 232: 2135–2144, 2017. © 2016 Wiley Periodicals, Inc. Lactate activates the LHR‐cAMP‐P450scc pathway to increase endogenous testosterone production. Treatment of CORT down‐regulates LHR‐cAMP‐PKA‐P450scc pathway to diminish testosterone production. Activation of intracellular adenylyl cyclase, PKA and P450scc pathways are critical to increase endogenous testosterone production in Leydig cells.