Two-pore channel 2 (TPC2) is a member of the endolysosomal ion channel family, playing critical roles in intracellular calcium signaling and endomembrane dynamics. This review provides an in-depth analysis of TPC2, covering its structural and functional properties, physiological roles, and involvement in human diseases. We discuss current experimental approaches to studying TPC2, including heterologous expression in plant vacuoles and computational modeling strategies. Particular emphasis is placed on the structural determinants of ion permeation, with a focus on the selectivity filter and the central cavity’s influence on channel kinetics. Furthermore, we explore emerging roles of TPC2 in viral infections, particularly SARS-CoV-2, and in cancer, including melanoma progression and neoangiogenesis. The inhibitory potential of natural compounds, such as naringenin, is also examined. By offering a comprehensive overview of current knowledge and methodologies, this review underscores the potential of TPC2 as a promising pharmacological target in both infectious and neoplastic diseases.