Objective To investigate the effect of nitric oxide (NO) on the scratching behavior evoked by substance P (SP) in mice. Methods Forty BALB/c mice were randomly divided into 5 groups to receive intradermal injection of different doses of SP (0, 20, 40, 80, 160 nmol/site) into the rostral part of the back to establish the acute itch model. Another 40 mice were randomly allocated to model, spantide, L-arginine,L-NAME and aminognanidine groups injected intracutaneously with normal saline (NS), spantide, L-arginine,L-NAME and aminoguanidine, respectively, 10 minutes before SP (80 nmol/site) injection. Subsequently,the scratching behavior was observed, iNOS expression and NO level in the injected skin were detected by immunohistochemical staining and nitrate reductase assay, respectively. Results The scratching bouts per hour induced by intradermal NS and SP of 20, 40, 80 and 160 nmol/site were 4.38±4.07, 5.38±3.78,12.75±6.52, 23.50±7.84 and 42.38±15.84, respectively, and only SP at higher doses (40 - 160 nmol/site)elicited a dose-dependent scratching response in mice (P < 0.01 or 0.05) compared with NS. The scratches over 1 hour in SP, L-arginine, spantide, L-NAME and aminognanidine group were 67.13±32.79, 70.75±34.80, 10.75±8.14, 29.00±21.19 and 35.38±22.83, respectively; of them, pretreatment with spantide,L-NAME and aminognanidine significantly inhibited SP-induced scratching (P < 0.01 or 0.05), but L-arginine showed no inhibitory effect (P > 0.05). Compared with SP, the pretreatment with spantide, L-NAME and aminoguanidine significantly downregulated the iNOS expression and NO content (P < 0.01 or 0.05) in the injected skin other than L-arginine (P > 0.05 ). Conclusion Intradermal SP could increase NO synthesis by neurokinin 1 receptor activation, resulting in the scratching behavior in BALB/c mice.