Cardiac dysfunction is a common complication in sepsis and is characterized by forward pump failure. Hallmarks of septic cardiomyopathy are decreased myofibrillar contractility and reduced Ca(2+) sensitivity but it is still not clear whether reduced pump efficiency is predominantly a diastolic impairment. Moreover, a comprehensive picture of upstream Ca(2+) handling mechanisms and downstream myosin biomechanical parameters is still missing. Ca(2+)-sensitizing agents in sepsis may be promising but mechanistic insights for drugs like levosimendan are scarce. Here, we used an endotoxemic LPS rat model to study mechanisms of sepsis on in vivo hemodynamics, multicellular myofibrillar Ca(2+) sensitivity, in vitro cellular Ca(2+) homeostasis and subcellular actomyosin interaction with intracardiac catheters, force transducers, confocal Fluo-4 Ca(2+) recordings in paced cardiomyocytes, and in vitro motility assay, respectively. Left ventricular ejection fraction and myofibrillar Ca(2+) sensitivity were depressed in LPS animals but restored by levosimendan. Diastolic Ca(2+) transient kinetics was slowed down by LPS but ameliorated by levosimendan. Selectively blocking intracellular and sarcolemmal Ca(2+) extrusion pathways revealed minor contribution of sarcoplasmic reticulum Ca(2+) ATPase (SERCA) to Ca(2+) transient diastole in LPS-evoked sepsis but rather depressed Na(+)/Ca(2+) exchanger and plasmalemmal Ca(2+) ATPase. This was mostly compensated by levosimendan. Actin sliding velocities were depressed in myosin heart extracts from LPS rats. We conclude that endotoxemia specifically impairs sarcolemmal diastolic Ca(2+) extrusion pathways resulting in intracellular diastolic Ca(2+) overload. Levosimendan, apart from stabilizing Ca(2+)-troponin C complexes, potently improves cellular Ca(2+) extrusion in the septic heart.