MiRNAs regulate the cardiomyocyte (CM) cell cycle at the post-transcriptional level, affect cell proliferation, and intervene in harmed CM repair post-injury. The present study was undertaken to characterize the role of let-7i-5p in the processes of CM cell cycle and proliferation and to reveal the mechanisms thereof. In the present study, we used real-time qPCR (RT-qPCR) to determine the up-regulated let-7i-5p in CMs during the postnatal switch from proliferation to terminal differentiation and further validated the role of let-7i-5p by loss- and gain-of-function of let-7i-5p in CMs in vitro and in vivo. We found that the overexpression of let-7i-5p inhibited CM proliferation, whereas the suppression of let-7i-5p significantly facilitated CM proliferation. E2F2 and CCND2 were identified as the targets of let-7i-5p, mediating its effect in regulating the cell cycle of CMs. Supperession of let-7i-5p promoted the recovery of heart function post-myocardial infarction by enhancing E2F2 and CCND2. Collectively, our results revealed that let-7i-5p is involved in the regulation of the CM cell cycle and further impacts proliferation, which may offer a new potential therapeutic strategy for cardiac repair after ischemic injury.