Cytocalasin D (cytoD) is a fungal metabolite that inhibits cytokinesis by blocking formation of contractile microfilament structures resulting in multi-nucleated cells, reversible inhibition of cell movement, and the induction of cellular extrusion. However, in muscle cells reports are controversial and vary from actin stabilization to actin disruption at concentrations of 40 mM. The aim of this study was to investigate the putative cytoD effects on adult cardiomyocytes in culture and how it can be used as a pharmacological tool in single cell models. This investigation is based on our optimized serum-free culture method for adult rat ventricular myocytes. Initially, for global calcium transients we performed repetitive dose response relationships over the time course of 6 days to identify the optimal cytoD concentration. At this identified concentration we analyzed action potentials, calcium handling (incl. post rest behavior and caffein induced calcium release) and cellular contractility. In addition, we investigated cytoD-mediated changes in the T-tubular system and actin cytoskeleton by confocal and STED microscopy, respectively. In contrast to previous reports we were able to identify a cytoD concentration that resulted in a high degree of morphological and functional conservation of properties found in freshly isolated cells over a time course of one week. In conclusion our findings represent a further significant improvement for single cell models of cardiomyocytes, enabling chronic stimulation and extended adenoviral protein expression. The identified relationship between actin filament modulation and prevention of T-tubular loss might shed new light on the loss of T-tubules in cardiac diseases.