Background and Purpose: Emerging evidence has shown that pinocembrin protects myocardial from ischemic injury in animals. However, it is unknown whether it has cardioprotection when given at the onset of reperfusion. Also, mechanisms mediating the cardioprotective actions of pinocembrin were largely unknown. Thus, this study aimed to investigate the effects of pinocembrin postconditioning on ischemia-reperfusion (I/R) injury and the underlying mechanisms. Experimental Approach: In vivo mouse model of myocardial I/R injury, ex vivo isolated rat heart with global I/R and in vitro hypoxia/reoxygenation (H/R) injury model for primary cardiomyocytes were used. Key Results: We found that pinocembrin postconditioning significantly reduced the infarct size and improved cardiac contractile function after acute myocardial I/R. Mechanically, in primary cardiomyocytes we found that pinocembrin may confer protection in part via direct stimulation of cardiac glycolysis via promoting the expression of glycolytic enzyme, PFKFB3. Besides, PFKFB3 inhibition abolished pinocembrin-induced glycolysis and protection in cardiomyocytes. More importantly, PFKFB3 knockdown via cardiotropic adeno-associated virus (AAV) abrogated cardioprotective effects of pinocembrin. Conclusions and Implications: In conclusion, these results established that the acute cardioprotective benefits of pinocembrin are mediated in part via glycolytic stimulation through PFKFB3, which may provide a new therapeutic target to impede the progression of myocardial I/R injury.