Circular RNAs are important players involving in a variety of physiological and pathological processes. However, their functions and mechanisms during myocardial ischemic injury and protection remain largely unknown. We recently found significant alterations of many circRNAs including circCHSY1 following myocardial ischemia/reperfusion (I/R) injury, whereas their exact functions are unclear. Here, we investigated roles of circCHSY1 in the acute myocardial I/R injury and the potential mechanisms involved. The expression of circCHSY1 was detected in cardiomyocytes from mouse, rat and human embryonic stem cells (hESC-CMs). It was further upregulated in mouse I/R (30 min/24 h) hearts, oxygen glucose deprivation/reperfused (OGD/R, 6 h/2 h) primary neonatal rat ventricular cardiomyocytes (NRCMs) and OGD/R (48 h/2 h) hESC-CMs. Adenovirus-mediated circCHSY1-overexpression significantly decreased infarct size and lactate dehydrogenase (LDH) release in mouse I/R hearts. Consistently, circCHSY1 overexpression reduced the LDH release in the OGD/R NRCMs and hESC-CMs, improved cell viability, and preserved mitochondrial function in the OGD/R NRCMs, whereas there were no significant differences in cell viability and LDH release between the OGD/R NRCMs with and without siRNA-mediated circCHSY1 knockdown. Mechanistically, circCHSY1 was detected to bind with miR-24-3p analyzed by dual luciferase assay and RNA pull-down assays. CircCHSY1 overexpression-mediated protective effects on cells and mitochondria in OGD/R NRCMs were reversed by the miR-24-3p mimic. Further, dual luciferase assay showed that miR-24-3p directly bound to heme oxygenase 1 (HO1) via its 3'UTR. The protein level of HO1 was downregulated by miR-24-3p mimic in OGD/R NRCMs but enhanced by the circCHSY1 overexpression in vitro and in vivo. Functionally, the HO1 knockdown by adenovirus in vivo and by siRNA in vitro eliminated cardioprotective effects of circCHSY1 overexpression. CircCHSY1 is upregulated following myocardial I/R injury. The higher level of circCHSY1 protects I/R hearts and cardiomyocytes. The protection of circCHSY1 is mediated through enhancement of the HO1 level, resulting in preserving mitochondrial homeostasis via targeting miR-24-3p in cardiomyocytes. These findings suggest circCHSY1 as a protective factor.