HIV‐1 Tat is essential for HIV‐1 replication and appears to play an important role in the pathogenesis of HIV‐associated neurological complications. Secreted from infected or transfected cells, Tat has the extraordinary ability to cross the plasma membrane. In the brain, Tat can be taken up by CNS cells via receptor‐mediated endocytosis. Following endocytosis and its internalization into endolysosomes, Tat must be released in order for it to activate the HIV‐1 LTR promoter and facilitate HIV‐1 viral replication in the nucleus. However, the underlying mechanisms whereby Tat escapes endolysosomes remain unclear. Because Tat disrupts intracellular calcium homeostasis, we investigated the involvement of calcium in Tat endolysosome escape and subsequent LTR transactivation. We demonstrated that chelating endolysosome calcium with high‐affinity rhodamine‐dextran or chelating cytosolic calcium with BAPTA‐AM attenuated Tat endolysosome escape and LTR transactivation. Significantly, we demonstrated that pharmacologically blocking and knocking down the endolysosome‐resident two‐pore channels (TPCs) attenuated Tat endolysosome escape and LTR transactivation. This calcium‐mediated effect appears to be selective for TPCs because knocking down TRPML1 calcium channels was without effect. Our findings suggest that calcium released from TPCs is involved in Tat endolysosome escape and subsequent LTR transactivation. TPCs might represent a novel therapeutic target against HIV‐1 infection and HIV‐associated neurological complications.