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Purpose: Anandamide, one of the endocannabinoids, has been reported to exhibit cardioprotective properties, particularly in its ability to limit the damage produced by ischemia reperfusion injury. However, the mechanisms underlying the effect are not well known. This study is to investigate whether anandamide alter Na+/Ca2+ exchanger and the intracellular free Ca2+ concentration ([Ca2+]i). Methods: Na+/Ca2+ exchanger current (INCX) was recorded and analysed by using whole-cell patch-clamp technique and [Ca2+]i was measured by loading myocytes with the fluorescent Ca2+ indicator Fura-2/AM. Results: We found that INCX was enhanced significantly after perfusion with simulated ischemic external solution; [Ca2+]i was also significantly increased by simulated ischemic solution. The reversal potential of INCX was shifted to negative potentials in simulated ischemic external solution. Anandamide (1–100 nM) failed to affect INCX and [Ca2+]i in normal solution. However, anandamide (1–100 nM) suppressed the increase in INCX in simulated ischemic external solution concentration-dependently and normalized INCX reversal potential. Furthermore, anandamide (100 nM) significantly attenuated the increase in [Ca2+]i in simulated ischemic solution. Blocking CB1 receptors with the specific antagonist AM251 (500 nM) failed to affect the effects of anandamide on INCX and [Ca2+]i in simulated ischemic solution. CB2 receptor antagonist AM630 (100 nM) eliminated the effects of anandamide on INCX and [Ca2+]i in simulated ischemic solution, and CB2 receptor agonist JWH133 (100 nM) simulated the effects of anandamide that suppressed the increase in INCX and [Ca2+]i in simulated ischemic solution. In addition, pretreatment with the Gi/o-specific inhibitor pertussis toxin (PTX, 500 ng/ml) eliminated the effects of anandamide and JWH133 on INCX in simulated ischemic solution. Conclusions: Collectively, these findings suggest that anandamide suppresses calcium overload through inhibition of INCX during perfusion with simulated ischemic solution; the effects may be mediated by CB2 receptor via PTX-sensitive Gi/o proteins. This mechanism is importantly involved in the anti-ischemia injury caused by endocannabinoids. [ABSTRACT FROM AUTHOR]