The myocardial protective effect of sevoflurane (Sev) has been confirmed, but its potential molecular mechanism remains not fully elucidated. To unravel the functional contribution and mechanistic basis of miR-300, its involvement in sevoflurane-elicited cardioprotection against myocardial hypoxia/reoxygenation (H/R) injury was probed in the present investigation. AC16 cardiomyocytes were preconditioned with Sev at concentrations of 0.5%, 1.0%, and 1.5%, followed by H/R treatment. miR‑300 and HMGB1 expression were quantified by qRT‑PCR. CCK-8 and flow cytometry were employed to evaluate cell proliferation and apoptosis, respectively. ELISA was used to quantify the expression of myocardial injury-related markers and inflammatory cytokine profiles. Commercial kits were applied to detect oxidative stress indicators (MDA, SOD, and CAT). miR‑300 directly targets HMGB1, as confirmed using dual‑luciferase reporter analysis. With the extension of hypoxia time, miR-300 expression in cardiomyocytes gradually decreased, and Sev preconditioning could inhibit this downregulation trend. Sev preconditioning significantly attenuated H/R-elicited loss of cellular viability, elevation of apoptotic levels, myocardial injury exacerbation, alongside inflammatory response generation and oxidative stress induction. Downregulation of miR-300 significantly weakened the myocardial protective effect of Sev. miR-300 could directly target HMGB1. HMGB1 knockdown abolished the impairment of Sev‑mediated cardioprotection caused by miR‑300 downregulation. miR-300 modulates the cardioprotective effect of sevoflurane preconditioning on myocardial injury by regulating HMGB1 expression, which may provide a new theoretical reference for the research of cardiovascular ischemia/reperfusion injury.