Chitooligosaccharide (COS) or oligochitosan has been shown to induce tobacco defense responses which are connected with nitric oxide (NO) and OIPK (oligochitosan-induced Ser/Thr protein kinase). The aim of this study was to reveal the relationship between NO production and OIPK pathway in the defense response of tobacco elicited by COS. NO generation was investigated by epidermal strip bioassay and fluorophore microscope using fluorophore diaminofluorescein diacetate (DAF-2DA). Tobacco epidermal cells treated with COS resulted in production of NO, which was first present in chloroplast, then in nucleus, finally in the whole cell; this NO production was sensitive to NO scavenger cPTIO and the mammalian NO synthase (NOS) inhibitor L: -NAME, suggesting that NOS-like enzyme maybe involved in NO generation in tobacco epidermal cells. However, NOS and nitrate reductase (NR, EC 1.6.6.1) inhibitors reduced NO content in tobacco leaves by using NO Assay Kit, suggesting both NOS and NR were involved in NO production in tobacco leaves. Using a pharmacological approach and western blotting, we provide evidence that NO acts upstream of OIPK expression. NO scavenger, NOS inhibitor partly blocked the activation of OIPK and the activities of several defense-related enzymes induced by COS; treatment with NO donor sodium nitroprusside (SNP) induced the activation of OIPK and enhanced the defense systems. The results suggest that COS is able to induce NO generation, which results in up-regulation the activities of some defense-related enzymes through an OIPK-dependent or independent pathway.