Ischemic preconditioning (PC) is a polygenic defensive cellular adaptive phenomenon whereby brief ischemic stimuli render the heart resistant to subsequent similar stress. The late phase of ischemic PC lasts for three to four days, protects against both myocardial stunning and infarction, and thus has considerable clinical relevance. Diverse signaling molecules released by a sublethal ischemic stress initiate a complex signal transduction cascade that modulates the expression of cardioprotective genes. Nitric oxide (NO), generated by the endothelial NO synthase (NOS) and acting via the formation of reactive oxygen species, activates the epsilon isoform of protein kinase C (PKC), which activates the Src family of protein tyrosine kinases (Src and Lck) and transcription factors (nuclear factor-kappaB, and possibly others), with resultant upregulation of the inducible NOS (iNOS) gene and protein expression. iNOS, and other cardioprotective proteins, including cyclooxygenase-2 and aldose reductase, confer resistance to subsequent ischemic stress. This delayed protection can also be mimicked, in the absence of ischemia, by administering NO-releasing agents, a situation that can be potentially exploited for cardioprotection in clinical situations. Identification of this novel bifunctional (trigger and mediator) role played by NO in cardiac protection, not only advances the knowledge regarding its signaling functions, but also offers a potential therapeutic strategy for patients with coronary artery disease. The purpose of this review is to summarize the current evidence in support of this critical role played by NO in ischemic PC.