AIMS: Mitochondrial oxidative stress (MOS) is a key contributor to poor cardiac function and a major driver of myocardial ischemia-reperfusion injury (MIRI). Our previous research demonstrated that stem cell-derived nanovesicles (NVs) enhanced cardiac function following ischemia-reperfusion (I/R) injury, although the underlying mechanisms remain unclear. We constructed and characterized miR-222-3p-loaded NVs. MATERIALS AND METHODS: An in vitro hypoxia-reoxygenation (H/R) model was established using H9C2 cardiomyocytes. Mitochondrial oxidative respiratory function was assessed using Seahorse XF technology, while mitochondrial reactive oxygen species (mtROS) levels were quantified via flow cytometry. Additional assessments included mitochondrial permeability transition pore (mPTP) status, mitochondrial membrane potential, and mitochondrial DNA (mtDNA) integrity. An in vivo H/R model was developed using C57BL/6 mice. The therapeutic effects of NVs on MOS reduction and cardiac function improvement were evaluated through Masson's staining, immunofluorescence, echocardiography, transmission electron microscopy (TEM), and positron emission tomography/computed tomography (PET/CT). KEY FINDINGS: RNA immunoprecipitation (RIP) confirmed that miR-222-3p directly targets cyp1a1. Overexpression of miR-222-3p or knockdown of cyp1a1 significantly improved mitochondrial activity in cardiomyocytes and conferred protection against I/R injury. Conversely, overexpression of cyp1a1 abrogated the protective effects of miR-222-3p. In vivo, NV treatment enhanced cardiac function, reduced MOS, and improved mitochondrial respiratory capacity in MIRI model mice. NV treatment, via miR-222-3p-mediated suppression of cyp1a1, mitigates MOS, enhances mitochondrial respiratory function, and improves cardiac outcomes in MIRI models. SIGNIFICANCE: These findings provide a foundational basis for the clinical translation of NV-based therapies.