Aim Since few studies have presented a thorough analysis of the effect of levosimendan (LEV) on contractility, our purpose was to investigate in vivo cardiac function as well as in vitro cardiomyocyte function and calcium (Ca2+) handling following LEV treatment. Methods Rats with post‐myocardial infarction heart failure (HF) induced by ligation of the left anterior descending coronary artery and sham‐operated animals were randomized to infusion of LEV (2.4 μg∙ kg−1∙ min−1) or vehicle for 40 min. Echocardiographic examination was coupled to pressure‐volume sampling in the left ventricle before (B) and after (40 min) infusion. Isolated left ventricular cardiomyocytes were studied in an epifluorescence microscope. Results HF LEV (n=6), HF vehicle (n=7), sham LEV (n=5) and sham vehicle (n=6) animals were included. LEV infusion compared to vehicle in HF animals reduced left ventricular end‐diastolic pressure and mean arterial pressure (both P<0.001) and improved the slope of the preload‐recruitable stroke work (P<0.05). Administrating LEV to HF cardiomyocytes in vitro improved fractional shortening and Ca2+ sensitivity index ratio, and increased the diastolic Ca2+ (all P<0.01). Conclusion In HF animals LEV improved the contractility by increasing the Ca2+ sensitivity. Furthermore loading conditions were changed, and LEV could consequently change organ perfusion. An observed increase in diastolic Ca2+ following LEV treatment and clinical implications of this should be further addressed. This article is protected by copyright. All rights reserved.