Objective To study the effect of NO donor (sodium nitroprusside, SNP) on testosterone secretion in rat Leydig cells. Methods The primary rat Leydig cells were cultured in vitro for 12 h, and then treated with different concentrations of SNP for various time courses (4 h, 8 h, and 12 h). The level of testosterone was detected by chemiluminescence, and the concentrations of 3β-hydroxysteroid dehydrogenase enzyme (3β-HSD) and annexin 5 were analyzed by Western blotting. Results After treatment with 10-4 mol/L SNP for 8 h, with 10-6 mol/L SNP for 12 h, or with 10-4 mol/L SNP for 12 h, the level of testosterone was increased by 23%, 28%, and 37% respectively, compared with that of the control (P <0.05). Western blot analysis showed that, when Leydig cells were stimulated by SNP, 3β-HSD expression had no significant difference (P >0.05), compared with that in the control. After Leydig cells were treated with different concentrations of SNP for 4 h or 8 h, the annexin 5 level also had no difference compared with that in the control (P >0.05). However, after Leydig cells were stimulated by 10-6 mol/L SNP for 12 h, the annexin 5 was increased by 55% compared with that of the control (P <0.05), and the expression of annexin 5 also increased by 48% in the cells treated with 10-4 mol/L SNP (P<0.05). Conclusions The results suggest the low concentration of SNP induces testosterone secretion in rat Leydig cells in a dose- and time-dependent manner. 3β-HSD is not involved in the process of testosterone secretion induced by SNP, but the process may be related to the expression of annexin 5.