Type I neuronal nitric oxide synthase (nNOS) neurons in neocortex are a subgroup of GABAergic neurons expressing both nNOS and substance P (SP) receptors (neurokinin 1, NK1 receptors). In the present study, we investigated the actions of SP on these neurons in acute slice preparations of visual cortex obtained from GAD67-GFP knock-in mice. Whole-cell patch clamp recording and Ca²⁺ imaging from type I nNOS neurons revealed that SP induced a Ca²⁺-permeable nonselective cation current. The SP-induced current was suppressed by ruthenium red, an inhibitor of transient receptor potential (TRP) channels. To clarify the signaling pathways following the activation of NK1 receptors, we examined the effects of inhibitors of intracellular signaling molecules. Among them, D609, an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), induced an outward current, and additional applications of SP induced an inward current which apparently reversed the D609-induced current. Both of these responses were not observed when Cs⁺-based intracellular solution was used, suggesting that they were mediated by K⁺ channels. In this recording condition, SP induced the TRP-like current which was markedly suppressed by D609. The present results suggest that SP activates TRP-like cation channels in type I nNOS neurons via a PC-PLC-dependent signaling, and inactivates some K⁺ channels which are usually inhibited by a PC-PLC-dependent mechanism.