Invariant natural killer T (iNKT) cells orchestrate tissue inflammation via regulating various cytokine productions. However the role of iNKT cells has not been determined in myocardial ischemia/reperfusion (I/R) injury. The purpose of this study was to examine whether the activation of iNKT cells by α-galactosylceramide (α-GC), which specifically activates iNKT cells, could affect myocardial I/R injury. I/R or sham operation was performed in male C57BL/6J mice. I/R mice received the injection of either αGC (I/R + αGC, n = 48) or vehicle (I/R + vehicle, n = 49) 30 min before reperfusion. After 24 h, infarct size/area at risk was smaller in I/R + αGC than in I/R + vehicle (37.8 ± 2.7% vs. 47.1 ± 2.5%, P < 0.05), with no significant changes in area at risk. The numbers of infiltrating myeloperoxidase- and CD3-positive cells were lower in I/R + αGC. Apoptosis evaluated by TUNEL staining and caspase-3 protein was also attenuated in I/R + αGC. Myocardial gene expression of tumor necrosis factor-α and interleukin (IL)-1β in I/R + αGC was lower to 46% and 80% of that in I/R + vehicle, respectively, whereas IL-10, IL-4, and interferon (IFN)-γ were higher in I/R + αGC than I/R + vehicle by 2.0, 4.1, and 9.6 folds, respectively. The administration of anti-IL-10 receptor antibody into I/R + αGC abolished the protective effects of αGC on I/R injury (infarct size/area at risk: 53.1 ± 5.2% vs. 37.4 ± 3.5%, P < 0.05). In contrast, anti-IL-4 and anti-IFN-γ antibodies did not exert such effects. In conclusion, activated iNKT cells by αGC play a protective role against myocardial I/R injury through the enhanced expression of IL-10. Therapies designed to activate iNKT cells might be beneficial to protect the heart from I/R injury.