Some organisms, such as adult zebrafish and newborn mice, have the capacity to regenerate heart tissue following injury. Unraveling the mechanisms of heart regeneration is fundamental to understanding why regeneration fails in adult humans. Numerous studies have revealed that nerves are crucial for organ regeneration, thus we aimed to determine whether nerves guide heart regeneration. Here, we show using transgenic zebrafish that inhibition of cardiac innervation leads to reduction of myocyte proliferation following injury. Specifically, pharmacological inhibition of cholinergic nerve function reduces cardiomyocyte proliferation in the injured hearts of both zebrafish and neonatal mice. Direct mechanical denervation impairs heart regeneration in neonatal mice, which was rescued by the administration of neuregulin 1 (NRG1) and nerve growth factor (NGF) recombinant proteins. Transcriptional analysis of mechanically denervated hearts revealed a blunted inflammatory and immune response following injury. These findings demonstrate that nerve function is required for both zebrafish and mouse heart regeneration. Display omitted •Hypo-innervation of the zebrafish heart impairs heart regeneration•Cholinergic signaling guides myocyte proliferation in zebrafish and neonatal mice•Neonatal mouse vagotomy impairs myocyte proliferation and heart regeneration•Neonatal vagotomy reduces the inflammatory response following heart injury Mahmoud et al. demonstrate that nerves are required for cardiomyocyte proliferation during both zebrafish and neonatal mouse heart regeneration. The negative effect of hypoinnervation on regeneration can be partially rescued by NRG1 and NGF proteins.