The present study aimed to investigate the inotropic effects of R(+)-pulegone, a monoterpene found in plant species belonging to the genus Mentha, on the mammalian heart. In electrically stimulated guinea pig atria, R(+)-pulegone reduced the contractile force (˜ 83%) and decreased the contraction time measured at 50% of the maximum force amplitude (CT50) from 45.8 ± 6.2 ms to 36.9 ± 6.2 ms, suggesting that R(+)-pulegone may have an effect on Ca2+ homeostasis. Nifedipine (40 μM), taken as a positive control, showed a very similar profile. To explore the hypothesis that R(+)-pulegone is somehow affecting Ca2+ handling, we determined concentration-response curves for both CaCl2 and BAY K8644. R(+)-pulegone shifted these curves rightward. Using isolated mouse ventricular cardiomyocytes, we measured whole-cell L-type Ca2+ current and observed an ICa,L peak reduction of 13.7 ± 2.5% and 40.2 ± 2.9% after a 3-min perfusion with 0.11 and 1.1 mM of R(+)-pulegone, respectively. In addition, the intracellular Ca2+ transient was decreased (72.9%) by 3.2 mM R(+)-pulegone, with no significant changes in [Ca2+]i transient decay kinetics. Moreover, R(+)-pulegone at 1.1 mM prolonged the action potential duration at 10, 50, and 90% of repolarisation. The lengthening of the action potential duration may be attributed to the substantial blockade of the outward K+ currents caused by 1.1 mM of R(+)-pulegone (90.5% at 60 mV). These findings suggest that R(+)-pulegone exerts its negative inotropic effect on mammalian heart mainly by decreasing the L-type Ca2+ current and the global intracellular Ca2+ transient.