The treatment of myocardial ischemia-reperfusion injury (IRI) requires urgent improvement of mitochondrial dysfunction and sustained energy supply to restore cardiac function, but currently, there is a lack of effective strategies to meet these needs. Here, we transplanted mitochondria to treat myocardial IRI by a sequential administration approach. First, nanomotors with chemotactic target ability are modified on the surface of mitochondria to obtain engineered mitochondrial nanomotors. Then, denatured bovine serum albumin is modified outside the nanomotor, enabling mitochondria to hitchhike on activated neutrophils to accumulate in the damaged heart. During reperfusion, immediate intramyocardial injection of these mitochondria can stabilize energy supply and rescue dying cardiomyocytes from IRI. In the subsequent tissue repair stage, the mitochondria injected intravenously can achieve stepwise targeting to the damaged heart by hitchhiking on activated neutrophils and chemotactic behavior of nanomotors, thereby continuously supplementing energy to cardiomyocytes and enhancing cardiac function. In addition, in vivo results show that sequential administration reduces adverse reactions such as arrhythmia caused by high-dose mitochondrial transplantation. Compared with existing treatment methods, this design of sequential administration is a special strategy targeting the specific needs and inflammatory microenvironment of myocardial IRI, better promoting the clinical translation of mitochondrial transplantation.