Cordycepin, a pure compound of Cordyceps sinensis (CS), is known as an adenosine analog. We have found that CS stimulated Leydig cell steroidogenesis. Here we investigated the in vivo and in vitro effects of cordycepin in primary mouse Leydig cell steroidogenesis. The results indicate that cordycepin increased the plasma testosterone concentration. Cordycepin also stimulated in vitro mouse Leydig cell testosterone production in dose- and time-dependent manners. We further observed that cordycepin regulated the mRNA expression of the A1, A2a, A2b, and A3 adenosine receptors in the mouse Leydig cells, and that antagonists of A1, A2a, and A3 suppressed testosterone production 20-50% testosterone production. Furthermore, Rp-cAMPS (cAMP antagonist) and Protein Kinase A (PKA) inhibitors (H89 and PKI) significantly decreased cordycepin-induced testosterone production, indicating that the PKA-cAMP signal pathway was activated by cordycepin through adenosine receptors. Moreover, cordycepin induced StAR protein expression, and H89 suppressed cordycepin-induced steroidogenic acute regulatory (StAR) protein expression. Conclusively, cordycepin associated with adenosine receptors to activate cAMP-PKA-StAR pathway and steroidogenesis in the mouse Leydig cells.