In the present study, the apoptotic effect of cordycepin on MA-10 cells, a mouse Leydig tumor cell line, was investigated. Results demonstrated that the number of rounding-up cell increased by cordycepin (10 μM to 5 mM for 24 h), and cells with plasma membrane blebbing could be observed by 100 μM cordycepin. In viability test, MA-10 cell surviving rate significantly decreased as the dosage (10 μM to 5 mM) and duration (3-24 h) of cordycepin treatment increased (P < 0.05). Cordycepin at 100 μM and 1 mM for 24 h treatment induced significant DNA fragmentation (P < 0.05). In addition, the percentage of G1 and G2/M phase cell significantly declined by cordycepin (100 μM and 1 mM) for 24 h treatment, while the percentages of subG1 phase cell increased by 100 μM and/or 1 mM cordycepin in 6, 12 and 24 h treatments (P < 0.05), respectively, which highly suggested that cordycepin induced MA-10 cell apoptosis. In mechanism study with the treatments of caspases, c-Jun NH(2) terminal kinase (JNK) or reactive oxygen species (ROS) inhibitors plus cordycepin for 24 h, only caspases inhibitor suppressed subG1 phase in MA-10 cells. Moreover, western blotting results showed that cordycepin induced caspase-9, -3 and -7 protein expressions, but not caspase-8, in time- and dose-dependent manners. In conclusion, cordycepin induced apoptosis in MA-10 mouse Leydig tumor cells through a caspase-9 and -3 and -7 dependent pathway.