Acidocalcisomes are acidic calcium and polyphosphate storage organelles found in a diverse range of organisms. Here we present evidence that the biogenesis of acidocalcisomes in Trypanosoma brucei is linked to the expression of adaptor protein-3 (AP-3) complex. Localization studies in cell lines expressing β3 and δ subunits of AP-3 fused to epitope tags revealed their partial co-localization with the vacuolar proton pyrophosphatase, a marker of acidocalcisomes, with the Golgi marker Golgi reassembly and stacking protein, and with antibodies against the small GTPase Rab11. Ablation of the β3 subunit by RNA interference (RNAi) resulted in disappearance of acidocalcisomes from both procyclic and bloodstream form trypanosomes, as revealed by immmunofluorescence and electron microscopy assays, with no alterations in trafficking of different markers to lysosomes. Knockdown of the β3 subunit resulted in lower acidic calcium, pyrophosphate, and polyphosphate content as well as defects in growth in culture, resistance to osmotic stress, and virulence in mice. Similar results were obtained by knocking down the expression of the δ subunit of AP-3. These results indicate that AP-3 is essential for the biogenesis of acidocalcisomes and for growth and virulence of T. brucei.