The neuropeptide nociceptin/orphanin FQ (N/OFQ) has been shown to inhibit delayed rectifier potassium current (IK) in acutely dissociated rat parietal cortical neurons. However, the detailed mechanism of N/OFQ-induced inhibition on IK is not clear. This study is the first to explore an involvement of calcium/calmodulin (CaM)-dependent protein kinase type II (CaMKII) in mediating N/OFQ-induced responses. Utilizing pharmacological inhibitors of CaM and CaMKII, we have investigated the contribution of CaMKII in N/OFQ-induced effects using the whole-cell patch-clamp technique. In whole-cell voltage clamp, W-7 (100 μM), an antagonist of CaM, as well as KN-62, an inhibitor of CaMKII activity, attenuated the inhibitory effects of N/OFQ on IK. Activation and inactivation analysis indicated that the kinetics of IK were altered by N/OFQ, with decreased activation and promoted inactivation of IK. W-7 and KN-62 (10 μM) partly abolished the activation and inactivation curves shift of IK induced by N/OFQ. These findings show that CaMKII plays a critical role in N/OFQ-induced inhibition of IK in acutely dissociated rat parietal cortical neurons.