Objective To observe the changes of mitochondria fusion protein 2 (Mfn2) and dynamin-related protein 1 (Drp1) in the cardioprotection of fasudil, and analyze the significance. Methods Hearts isolated from male Sprague-Dawley rats were subjected to ischemia for 30 minutes (occlusion of left anterior descending artery), and continuously perfusion for 120 minutes to establish myocardial ischemia/reperfusion (I/R) injury model. The rats were divided into 3 groups: sham group, I/R group and fasudil group. The left ventricular hemodynamics were continuously recorded; lactate dehydrogenase (LDH) content was measured during reperfusion; myocardial ultrastructure was observed by electron microscopy; the protein expression of phosphorylated protein phosphatase 1 regulatory subunit 12A (p-PPP1R12A/p-MYPT1) was detected by immunohistochemistry; and the protein expressions of Mfn2, Drp1 and cleaved caspase-3 (c-caspase-3) were detected by Western blot analysis. Results Compared with the sham group, the left ventricular systolic and diastolic function was weakened, and LDH release was promoted in the other two groups during reperfusion. Compared with the I/R group, fasudil improved left ventricular systolic and diastolic function and reduced LDH release. Electron microscopy and immunohistochemical results showed that myofibril and mitochondria were damaged obviously, and p-MYPT1 protein expression was enhanced in the I/R group. Compared with the I/R group, fasudil attenuated the damage of myofibril and mitochondria, and decreased p-MYPT1 protein expression. Western blotting showed that, compared with the sham group, Mfn2 protein expression decreased, Drp1 and c-caspase-3 protein expressions increased in the I/R group. Compared with I/R group, there was no obvious change in Mfn2 protein expression, while Drp1 and c-caspase 3 protein expressions decreased in the fasudil group. Conclusion Fasudil can protect against myocardial I/R damage through inhibiting Rho kinase, which has no clear correlation with Mfn2 protein expression, but may be related with decreasing Drp1 protein expression and reducing mitochondrial injury, thereby inhibiting apoptosis.