This review explores the roles of the cholinergic system in the heart, comprising the neuronal and non-neuronal cholinergic systems. Both systems are essential for maintaining cardiac homeostasis by regulating the release of acetylcholine (ACh). A reduction in ACh release is associated with the early onset of cardiovascular diseases (CVDs), and increasing evidence supports the protective roles of ACh against CVD. We address the challenges and limitations of current strategies to elevate ACh levels, including vagus nerve stimulation and pharmacological interventions such as cholinesterase inhibitors. Additionally, we introduce alternative strategies to increase ACh in the heart, such as stem cell therapy, gene therapy, microRNAs, and nanoparticle drug delivery methods. These findings offer new insights into advanced treatments for regenerating the injured human heart.