Whereas prior studies have demonstrated an important immunomodulatory role for the neuronal cholinergic system in the heart, the role of the nonneuronal cholinergic system is not well understood. To address the immunomodulatory role of the nonneuronal cholinergic system in the heart, we used a previously validated diphtheria toxin–induced (DT-induced) cardiomyocyte ablation model (Rosa26-DTMlc2v-Cre mice). DT-injected Rosa26-DTMlc2v-Cre mice were treated with diluent or pyridostigmine bromide (PYR), a reversible cholinesterase inhibitor. PYR treatment resulted in increased survival and decreased numbers of MHC-IIloCCR2+ macrophages in DT-injected Rosa26-DTMlc2v-Cre mice compared with diluent-treated Rosa26-DTMlc2v-Cre mice. Importantly, the expression of CCL2/7 mRNA and protein was reduced in the hearts of PYR-treated mice. Backcrossing Rosa26-DTMlc2v-Cre mice with a transgenic mouse line (Chat-ChR2) that constitutively overexpresses the vesicular acetylcholine transporter (VAChT) resulted in decreased expression of Ccl2/7 mRNA and decreased numbers of CD68+ cells in DT-injured Rosa26-DTMlc2v-Cre/Chat-ChR2 mouse hearts, consistent with the pharmacologic studies with PYR. In vitro studies with cultures of LPS-stimulated peritoneal macrophages revealed a concentration-dependent reduction in CCL2 secretion following stimulation with acetylcholine, nicotine, and muscarine. To our knowledge, these findings reveal a previously unappreciated immunomodulatory role for the nonneuronal cholinergic system in regulating homeostatic responses in the heart following tissue injury.