MicroRNA-335 (miR-335) is implicated in several pathophysiological processes, including tumorigenesis, lipid metabolism and ischemic stroke; however, whether miR-335 plays a role in modulating myocardial ischemia reperfusion injury (MIRI) is still unknown. This study is aimed to explore the role and mechanism of miR-335 in the pathophysiological process of MIRI. Specifically, miR-335 mimics or a chemically modified agomiR-335 were transfected or injected into H9c2 cells and Wistar rats to upregulate miR-335 expression in vitro and in vivo, respectively. The effects of miR-335 overexpression on hypoxia/reoxygenation (H/R)-treated cardiomyocytes and ischemia/reperfusion (I/R)-exposed heart samples were investigated by a Cell Counting Kit-8 assay, flow cytometry, TTC staining and a TUNEL assay. The target of miR-335 was identified using a luciferase reporter assay. The expression of heme oxygenase 1 (HO-1) and inducible nitric oxide synthase (iNOS) was detected by reverse transcription-quantitative polymerase chain reaction and western blotting. The results showed that miR-335 expression in cardiomyocytes and the myocardium was downregulated during MIRI but was induced by hypoxic/ischemic postconditioning. MiR-335 overexpression led to an increase in cell viability and a reduction in the apoptosis of H/R-treated cardiomyocytes. Meanwhile, myocardial infarct size and the apoptosis of I/R-exposed heart tissues were decreased in response to miR-335 upregulation. Furthermore, we identified that hypoxia inducible factor 1-alpha subunit inhibitor (HIF1AN), a suppressor of hypoxia inducible factor 1-alpha (HIF-1α) stabilization and transcriptional activity, is a novel target of miR-335. MiR-335 overexpression enhanced the transcriptional activity of HIF-1α, increased the expression of HO-1 and iNOS, and inhibited mitochondrial permeability transition pore (MPTP) opening. In conclusion, we are the first to demonstrate that upregulation of miR-335 ameliorates MIRI by targeting HIF1AN. Thus, miR-335 may be a new therapeutic target for the treatment of MIRI.