Anesthetics are used to produce hypnosis and analgesic effects during surgery, but anesthesia for a long time after the operation is not conducive to the recovery of animals or patients. Therefore, finding appropriate treatments to counter the effects of anesthetics could enhance postoperative recovery. In the current study, we discovered the novel role of a GluN2A-selective positive allosteric modulator (PAM) in ketamine-induced anesthesia and investigated the effects of the PAM combined with nalmefene and flumazenil (PNF) in reversing the actions of an anesthetic combination (ketamine-fentanyl-dexmedetomidine, KFD). PAM treatment dose-dependently decreased the duration of the ketamine-induced loss of righting reflex (LORR). Compared with those in the KFD group, the duration of LORR and the analgesic effect of the KFD + PNF group were obviously decreased. Meanwhile, successive administration of PNF and KFD had no adverse effects on the cardiovascular and respiratory systems. Both the KFD group and the KFD + PNF group showed no changes in hepatic and renal function or cognitive function in rats. Moreover, the recovery of motor coordination of the KFD + PNF group was faster than that of the KFD group. In summary, our results suggest the potential application of the PNF combination as an antagonistic treatment strategy for anesthesia.