Haloperidol is a typical antipsychotic drug with inhibitory effects on dopamine and calcium homeostasis. In this study, the effect of haloperidol on the inotropism of rabbits’ isolated heart was investigated by measuring the isovolumetric left ventricular pressure using a balloon in a modified Langendorff perfusion apparatus. Haloperidol at 0.01–0.3 µmol/l induced a negative inotropic effect (Emax = 77.95 ± 0.19; EC50 = 0.043 ± 0.002 µmol/l). The effect of haloperidol was decreased by Ca2+ (Emax = 42.93 ± 3.22; EC50 = 0.37 ± 0.07 µmol/l; pD’2 = 7.01 ± 0.16), Bay K 8644 (Emax = 30.75 ± 1.33; EC50 = 10.43 ± 1.5 µmol/l, pD’2 = 7.13 ± 0.12), and digoxin (Emax = 42.03 ± 3.72, EC50 = 0.32 ± 0.05 µmol/l, pD’2 = 6.81 ± 0.14). The effect of haloperidol was also reduced by norepinephrine (Emax = 37.16 ± 1.84; EC50 = 1.73 ± 0.24 µmol/l, pD’2 = 6.97 ± 0.08) and dopamine (Emax = 35.68 ± 2.78; EC50 = 0.69 ± 0.01 µmol/l, pD’2 7.48 ± 0.15). However, the effect of haloperidol was nonsignificantly reduced by dobutamine (Emax = 58.89 ± 5.18; EC50 = 0.15 ± 0.06 µmol/l, pD’2 = 5.88 ± 0.47). These results show that the drugs that increase the influx of Ca2+ into the cardiomyocyte decrease the negative inotropic effect of haloperidol, suggesting that the effect of haloperidol could be mediated via mechanisms involving actions on Ca2+ entry into the cardiomyocyte. Haloperidol should be used carefully when prescribed for patients with cardiovascular disorders.