The activation of hypoxia-inducible factor (HIF) is an important event for mediating the adaptive response to myocardial ischemia/reperfusion (MI/R) injury. The ubiquitin-conjugating enzyme E2S (Ube2s) catalyzes ubiquitin conjugation to target proteins. Here, we report the positive regulation of HIF-1α signaling by Ube2s via stabilizing β-catenin, by which Ube2s acts to protect against MI/R injury. We show that Ube2s expression is upregulated in the hearts of mice subjected to MI/R injury. Functionally, Ube2s depletion exacerbates and its overexpression ameliorates MI/R injury. In addition, Ube2s augments the activation of HIF-1α and reduces myocardial apoptosis. Moreover, Ube2s induces the accumulation of β-Catenin through increasing its stabilization. Importantly, β-Catenin knockdown abrogates Ube2s-augmented HIF-1α activation, and meanwhile, diminishes the protective effect of Ube2s on MI/R injury, thus establishing a causal link between Ube2s-stabilized β-catenin and HIF-1α-mediated myocardial protection. Altogether, this study identifies the Ube2s/β-catenin/HIF-1α axis as a novel protective regulator involved in MI/R injury, and also implies that it might represent a potential therapeutic target for ameliorating MI/R injury.