Acute myocardial infarction due to ischemia/reperfusion (I/R) injury remains a major global health burden. Ischemic preconditioning (IPC)—brief cycles of I/R before prolonged ischemia—and vagal nerve activation confer with the onset of reperfusion cardioprotection via endogenous signaling pathways. Acetylcholine (ACh), released from the intracardiac nervous system (ICNS) upon vagal stimulation, is considered to be cardioprotective. Recent studies suggest cardiomyocytes can also synthesize and release ACh via a non-neuronal cholinergic cardiac system (NNCCS), independent of neuronal excitation, potentially acting as an autocrine/paracrine mediator of protection. This thesis investigates in a reductionist approach if hypoxic preconditioning (HPC), as a surrogate for IPC, induces protection in isolated adult rat ventricular cardiomyocytes against subsequent hypoxia/reoxygenation (H/R) injury, representing myocardial I/R injury. Cardiomyocyte viability was assessed before and after H/R and potential underlying signaling pathways were characterized in this way using pharmacological blockers for autacoid receptors and cardioprotective intracellular pathways: reperfusion injury salvage kinase (RISK) pathway, survivor activating factor enhancement (SAFE) pathway, and protein kinase C (PKC). Intra- and extracellular ACh concentrations were quantified by mass spectrometry. The dependence of protection through HPC, or through pharmacological conditioning (in order to increase ACh availability, using the ACh analogue carbachol or the ACh esterase inhibitor physostigmine) on muscarinic and nicotinic ACh receptor activation was examined using respective pharmacological receptor blockers. HPC preserved cardiomyocyte viability following H/R. Blocking autacoid receptors, RISK pathway, or PKC abrogated HPC’s protective effect, confirming HPC as a valid IPC surrogate. HPC increased intra- and extracellular ACh during hypoxia, demonstrating NNCCS activation. Blocking muscarinic and nicotinic receptors during H/R abolished protection through HPC. Pharmacological conditioning also conferred similar receptor-dependent protection. In conclusion, this thesis provides first-time evidence that HPC induces cardiomyocyte protection via NNCCS activation, with endogenous non-neuronal ACh acting as an autocrine/paracrine trigger, independently of the ICNS. Understanding this cholinergic mechanism offers new insights into potential therapeutic targets for mitigating myocardial I/R injury through cardioprotective interventions.