To research the effect of insulin on the expression and activity of the neuronal nitric oxide synthase in neural cells, the effects of insulin on the expression and activity of neuronal nitric oxide synthase were investigated in PC12 cells, by using flow cytometry, in situ hybridization and electron spin resonance (ESR) techniques. After incubation with insulin for 9 h, the expression of nNOS protein was significantly increased by insulin in a concentration-dependent manner, with a maximal increase of about 55% of the control values (P＜0.01, n=3, t-test). The transcription of nNOS was also significantly increased by insulin (16 mU/L, 6 h), reaching （182±13）% of the control values (P＜0.01, n=3, t-test). In addition, significant increase of the activity of nNOS in PC12 cells was also detected after incubation with insulin (16 mU/L) for 9 h, with an increase of 67% of the control values (P＜0.01, n=4, t-test). It can be concluded that insulin can upregulate the expression and activity of neuronal nitric oxide synthase in PC12 cells.