The aim of the present study was to investigate the effects of betulinic acid (BetA) on the expression and distribution pattern of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), an indirect indicator of nitric oxide (NO) synthase in the thymus and spleen of mice. Mice were randomly assigned to four main groups (n=48 per group): Experimental group (BetA), positive control group (goniothalamin), vehicle control group (dimethyl sulfoxide) and control group (without vehicle). Each group was further divided into three equal subgroups according to the treatment length (4, 8 and 12 days). BetA treatment induced the expression of NADPH-d activity in the thymus and spleen without any significant changes in the morphology of the organs. Furthermore, the expression pattern of NADPH-d in BetA-treated animals was significantly increased compared with that in the control animals. NADPH-d expression in the thymus and spleen suggests that NO signaling may be a potential mechanism underlying the BetA-induced immunomodulation in these organs. These findings are of direct clinical relevance and may contribute to the further development of BetA as a therapeutic drug.