Drosophila harbours a simple tubular heart that ensures haemolymph circulation within the body. The heart is built by a few different cell types, including cardiomyocytes that define the luminal heart channel and ostia cells that constitute openings in the heart wall allowing haemolymph to enter the heart chamber. Regulation of flow directionality within a tube, such as blood flow in arteries or insect haemolymph within the heart lumen, requires a dedicated gate, valve or flap-like structure that prevents backflow of fluids. In the Drosophila heart, intracardiac valves provide this directionality of haemolymph streaming, with one valve being present in larvae and three valves in the adult fly. Each valve is built by two specialised cardiomyocytes that exhibit a unique histology. We found that the capacity to open and close the heart lumen relies on a unique myofibrillar setting as well as on the presence of large membranous vesicles. These vesicles are of endocytic origin and probably represent unique organelles of valve cells. Moreover, we characterised the working mode of the cells in real time. Valve cells exhibit a highly flexible shape and, during each heartbeat, oscillating shape changes result in closing and opening of the heart channel. Finally, we identified a set of novel valve cell markers useful for future in-depth analyses of cell differentiation in wild-type and mutant animals.