The aim of this study was to investigate the protective effects of vitamin D (VD) against myocardial ischemia-reperfusion (I/R) injury in hearts. An I/R injury model was induced by left coronary artery ligation in Sprague-Dawley rats (in vivo) and Langendorff perfusion of isolated hearts (in vitro). The infarction areas were determined by triphenyltetrazolium chloride (TTC) staining. Changes in the ST segment, cardiac function, lactate dehydrogenase (LDH) activity, creatine kinase (CK) activity, inflammatory cytokine (interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α)) levels and the RhoA/ROCK/NF-ĸB pathway were tested in rats with I/R injury treated with or without VD. VD notably alleviated myocardial injury with decreased infarction areas and had a restorative effect on cardiac function, which was specifically manifested as a restored ST segment, increased myocardial contractility and increased coronary blood flow in the isolated hearts. The levels of CK and LDH were also suppressed by VD. In addition, VD significantly decreased the expression of inflammatory cytokines in rat sera and isolated hearts. The RhoA/ROCK/NF-κB pathway in I/R-injured rats was also obviously inhibited with VD treatment. The present study demonstrates that VD plays a protective role against myocardial injury by inhibiting inflammation through repressing the RhoA/ROCK/NF-κB pathway.