The aim of this study was to test whether morphine prevents the mitochondrial permeability transition pore (mPTP) opening through Zn2+ and glycogen synthase kinase 3β (GSK-3β). Fluorescence dyes including Newport Green Dichlorofluorescein (DCF), 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM), and tetramethylrhodamine ethyl ester (TMRE) were used to image free Zn2+, nitric oxide (NO), and mitochondrial membrane potential (ΔΨm), respectively. Fluorescence images were obtained with confocal microscopy. Cardiomyocytes treated with morphine for 10 min showed a significant increase in Newport Green DCF fluorescence intensity, an effect that was reversed by the NO synthase inhibitor N G-nitro-l-arginine methyl ester (l-NAME), indicating that morphine mobilizes Zn2+ via NO. Morphine rapidly produced NO. ODQ and NS2028, the inhibitors of guanylyl cyclase, prevented Zn2+ release by morphine, implying that cGMP is involved in the action of morphine. The effect of morphine on Zn2+ release was also abolished by KT5823, a specific inhibitor of protein kinase G (PKG). Morphine prevented oxidant-induced loss of ΔΨm, indicating that morphine can modulate the mPTP opening. The effect of morphine on the mPTP was reversed by KT5823 and the Zn2+ chelator N, N, N′, N′-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN). The action of morphine on the mPTP was lost in cells transfected with the constitutively active GSK-3β mutant, suggesting that morphine may prevent the mPTP opening by inactivating GSK-3β. In support, morphine significantly enhanced phosphorylation of GSK-3β at Ser9, and this was blocked by TPEN. GSK-3β small interfering RNA prevented the pore opening in the control cardiomyocytes but failed to enhance the effect of morphine on the mPTP opening. In conclusion, morphine mobilizes intracellular Zn2+ through the NO/cGMP/PKG signaling pathway and prevents the mPTP opening by inactivating GSK-3β through Zn2+.