target of rapamycin complex 1; polyP, polyphosphate; PPi, pyrophosphate; PtdIns3K, phosphatidylinositol 3-kinase; PtdIns3P, phosphatidylinositol 3-phosphate; RNAi, RNA interference; T. brucei, Trypanosoma brucei; Tbb3, the b3 subunit of adaptor protein-3 complex in Trypanosoma brucei; Tbd, the d subunit of adaptor protein-3 complex in Trypanosoma brucei; TbVMA1, the subunit A of V-H C -ATPase in Trypanosoma brucei; TbVP1, vacuolar pyrophosphatase in Trypanosoma brucei; TbVPH1, the a subunit of V-H C -ATPase in Trypanosoma brucei; TOR, target of rapamycin; V-H C -ATPase, vacuolar-type H C -ATPase; V-PPase, vacuolar pyrophophatase.Lysosomes play important roles in autophagy, not only in autophagosome degradation, but also in autophagy initiation. In Trypanosoma brucei, an early divergent protozoan parasite, we discovered a previously unappreciated function of the acidocalcisome, a lysosome-related organelle characterized by acidic pH and large content of Ca 2C and polyphosphates, in autophagy regulation. Starvation-and chemical-induced autophagy is accompanied with acidocalcisome acidification, and blocking the acidification completely inhibits autophagosome formation. Blocking acidocalcisome biogenesis by depleting the adaptor protein-3 complex, which does not affect lysosome biogenesis or function, also inhibits autophagy. Overall, our results support the role of the acidocalcisome, a conserved organelle from bacteria to human, as a relevant regulator in autophagy.