Objective To investigate the effect of miR-29a interference on mitochondrial fusion and fission of cardiomyocytes induced by oxygen and glucose deprivation/reoxygenation (OGD/R). Methods H9c2 cells were divided into normal control group, model group, negative control group and miR-29a interference group. Rat H9c2 cardiomyocyte injury model was induced by OGD/R. Negative control (NC) group cells were transfected with anti-NC, while miR-29a interference group cells were transfected with anti-miR-29a, and normal control group cells were not transfected. Reverse transcription PCR was used to detect miR-29a interference efficiency, along with CCK-8 assay to detect cell proliferation ratio, flow cytometry to detect cell apoptosis rate and mitochondrial membrane potential change, and kit to detect superoxide dismutase(SOD), malondialdehyde (MDA), lactate dehydrogenase (LDH)contents. Western blot analysis was employed to test the levels of Bcl2-associated X protein (BAX), Bcl2 antagonist/killer (BAK), caspase-9, mitochondrial fission protein 1(Fis1), mitofusin 1 (Mfn1), Mfn2, optic atrophy 1(OPA1), phosphoryerated extracellular signal-regulated kinase (p-ERK) and phosphoryerated dynamin related protein-1(p-Drp1). Results Compared with OGD/R group, the expression level of miR-29a of H9c2 cells in OGD/R group treated with anti-miR-29a decreased significantly, together with the findings including significantly increased cell proliferation factor, decreased apoptosis rate, increased SOD content, decreased MDA and LDH contents, as well as significantly increased mitochondrial membrane potential. The protein levels of BAX, BAK, caspase-9, Fis1, Mfn1, Mfn2, OPA1, p-ERK and p-Drp1 significantly decreased. Conclusion Interference with miR-29a expression can promote OGD/R-induced proliferation of H9c2 cells, inhibit cell apoptosis, reduce mitochondrial oxidative stress level, enhance mitochondrial membrane potential, and alleviate mitochondrial over-fusion and fission of myocardial cells.