The calcium ion (Ca²⁺) plays a fundamental role in the metabolism and cell physiology of eukaryotic cells. In general, increases in cytosolic Ca²⁺ may come from both of the extracellular environment through specific channels and/or calcium release from intracellular stores. The mechanism by which the ion calcium (Ca²⁺) is released from intracellular stores in higher eukaryotes is well known; however, in lower eukaryotes is still a subject of study. In the present work, it was elucidated that Trypanosoma cruzi epimastigotes can release Ca²⁺ from intracellular stores in response to high osmolarity, in a process involving a protein kinase-regulated Na⁺/H⁺ exchanger present in the acidocalsisomes of the parasite. In addition, we demonstrated that epimastigote membranes are able to release Ca²⁺ in response to exogenous activators of both inositol 1,4,5-triphosphate (IP₃) and Ryanodine receptors. Furthermore, we also summarize the involvement of calcium-related signaling pathways in biochemical and morphological changes triggered by hyperosmotic stress in T. cruzi epimastigotes.