Background: Intercellular interactions between cardiomyocytes (CMs) and cardiofibroblasts (FBs) are important in the physiological and pathophysiological heart. Understanding such interactions is important for developing effective therapies for heart disease. However, little is known about these interactions until recently. Membrane nanotube (MNT) is a novel structure connecting the mammalian cells over long distance. The classical view is that paracrine and gap junctions are the only forms of “communication” between MCs and FBs. Methods and Results: By membrane dye staining, we observed long, thin membrane nanotubular structures containing actin and microtubules that connected neonatal rat ventricular CMs and FBs. The mean length of WGA-stained membrane nanotubes between CMs and FBs was 13.9±10.4 mm (n=73 of 39 pairs of cells). By single-particle tracking, we observed vehicles moving between CMs and FBs within the membrane nanotubes. By dual colour staining, confocal imaging and flow cytometry, we observed mitochondria exchange between CMs and FBs in a coculture system. By combined atomic force microscopy (AFM) and confocal microscopy, we observed calcium signal propagation from AFM-stimulated CM or FB to unstimulated FB or CM via membrane nanotubes. By membrane and cytoskeleton staining, we observed similar nanotubular structures in adult mouse heart tissue, which suggests their physiological relevance. Conclusion: Our study posts a challenge to the classical “cardiac communication” theory, and suggests that MNT is an important structure for long distance “communication” between cardiac cells.