It was investigated whether the effects of sinusoidal electromagnetic field (SEMF) stimulation on the differentiation and mineralization of osteoblast are mediated by the nitric oxide (NO) signal pathway. It was first investigated whether SEMF had an effect on nitric oxide synthase (NOS) activity measured on the 0 h, 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h after SEMF treatment. Secondly, the NOS inhibitor, L-nitro-arginine-methylester (L-NAME) was added to the culture medium to observe whether it inhibit the maturation and mineralization of osteoblast stimulated by SEMF, which evaluated by measuring alkaline phosphatase (ALP) activity, CFU-F_ALP, osterix gene expression and mineralized bone modulus. After treatment of SEMF, the NOS activity in SEMF group increased in comparison with the normal control group (P < 0.01), reaching the highest level after 0.5 h. The gene expression of Osterix, ALPase activity and mineralized bone modulus in the SEMF group were also increased significantly. However, these effects were partially blocked in the L-NAME group．Surprisingly, all the osteogenic markers in the SEMF+L-NAME group were slightly higher than those of the control group, but lower than those of the SEMF group．In conclusion, The NO signal pathway was activated by SEMF treatment．The stimulatory effects of SEMF on the differentiation and mineralization of osteoblast were attenuated when NO signal pathway was blocked.