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microRNA-210 (miR-210) is upregulated in hypoxia, but its function in cardiomyocytes and its regulation in response to hypoxia are not well characterized. The purpose of this study was to identify upstream regulators of miR-210, as well as to characterize miR-210's function in cardiomyocytes. We first showed miR-210 is upregulated through both hypoxia-inducible factor (HIF)-dependent and -independent pathways, since aryl hydrocarbon nuclear translocator (ARNT) knockout mouse embryonic fibroblasts (MEF), lacking intact HIF signaling, still displayed increased miR-210 levels in hypoxia. To determine the mechanism for HIF-independent regulation of miR-210, we focused on p53 and protein kinase B (Akt). Overexpression of p53 in wild-type MEFs induced miR-210, whereas p53 overexpression in ARNT knockout MEFs did not, suggesting p53 regulates miR-210 in a HIF-dependent mechanism. Akt inhibition reduced miR-210 induction by hypoxia, whereas Akt overexpression increased miR-210 levels in both wild-type and ARNT knockout MEFs, indicating Akt regulation of miR-210 is HIF-independent. We then studied the effects of miR-210 in cardiomyocytes. Overexpression of miR-210 reduced cell death in response to oxidative stress and reduced reactive oxygen species (ROS) production both at baseline and after treatment with antimycin A. Furthermore, downregulation of miR-210 increased ROS after hypoxia-reoxygenation. To determine a mechanism for the cytoprotective effects of miR-210, we focused on the predicted target, apoptosis-inducing factor, mitochondrion-associated 3 (AIFM3), known to induce cell death. Although miR-210 reduced AIFM3 levels, overexpression of AIFM3 in the presence of miR-210 overexpression did not reduce cellular viability either at baseline or after hydrogen peroxide treatment, suggesting AIFM3 does not mediate miR-210's cytoprotective effects. Furthermore, HIF-3α, a negative regulator of HIF signaling, is targeted by miR-210, but miR-210 does not modulate HIF activity. In conclusion, we demonstrate a novel role for p53 and Akt in regulating miR-210 and demonstrate that, in cardiomyocytes, miR-210 exerts cytoprotective effects, potentially by reducing mitochondrial ROS production.