Nitric oxide (NO), a vasoactive gas that can freely diffuse into the cell, has many physiological effects in various cell types. Since 1986, numerous studies of ischemic preconditioning against ischemia-reperfusion (I/R) injury have been undertaken and the roles of the NO signaling pathway in cardioprotection have been explored. Many studies have confirmed the effect of NO and that its relative signaling pathway is important for preconditioning of the cardioprotective effect. The NO signaling against I/R injury targeted on the mitochondria is believed to be the end-target for cardioprotection. If the NO signaling pathway is disrupted or inhibited, cardioprotection by preconditioning disappears. During preconditioning, signaling is initiated from the sarcolemmal membrane, and then spread into the cytoplasm via many series of enzymes, including nitric oxide synthase (NOS), the NO-producing enzyme, soluble guanylyl cyclase (sGC), and protein kinase G (PKG). Finally, the signal is transmitted into the mitochondria, where the cardioprotective effect occurs. It is now well established that mitochondria act to protect the heart against I/R injury via the opening of the mitochondrial ATP-sensitive K+ channel and the inhibition of mitochondrial permeability transition (MPT). This knowledge may be useful in developing novel strategies for clinical cardioprotection from I/R injury. © Med Sci Monit.