AbstractCardiomyocytes derived from human induced pluripotent stem cells (hiPSC‐CMs) have the potential to be a therapeutic option for myocardium restoration. However, hiPSC‐CMs of varying maturation and transplantation routes exhibit different reactivity and therapeutic effects. We previously demonstrated that the saponin+compound induces more mature hiPSC‐CMs. The safety and efficacy of multi‐route transplantation of saponin+compound‐induced hiPSC‐CMs in a nonhuman primate with myocardial infarction will be investigated for the first time in this study. Our findings indicate that optimized hiPSC‐CMs transplanted via intramyocardial and intravenous routes may affect myocardial functions by homing or mitochondrial transfer to the damaged myocardium to play a direct therapeutic role as well as indirect beneficial roles via anti‐apoptotic and pro‐angiogenesis mechanisms mediated by different paracrine growth factors. Due to significant mural thrombosis, higher mortality, and unilateral renal shrinkage, intracoronary transplantation of hiPSC‐CMs requires closer attention to anticoagulation and caution in clinical use. Collectively, our data strongly indicated that intramyocardial transplantation of hiPSC‐CMs is the ideal technique for clinical application; multiple cell transfers are recommended to achieve steady and protracted efficacy because intravenous transplantation's potency fluctuates. Thus, our study offers a rationale for choosing a therapeutic cell therapy and the best transplantation strategy for optimally induced hiPSC‐CMs.