Trypanosoma brucei and T. cruzi are the etiologic agents of the most important human trypanosomal diseases: Sleeping sickness (human African trypanosomiasis) and Chagas disease (American trypanosomiasis). Both diseases are potentially fatal and there are no vaccines or satisfactory treatments available for them. We are interested in investigating proteins that can be used as chemotherapeutic targets to develop efficient treatments against African and American trypanosomiasis. We are particularly interested in establishing the role of proteins involved in polyphosphate (polyP) metabolism. PolyP is an anionic polymer of orthophosphate groups linked by high-energy bonds that typically accumulates in acidic, calcium-rich organelles known as acidocalcisomes. PolyP synthesis in eukaryotes was unclear until recent work demonstrated that the protein named vacuolar transporter chaperone 4 (Vtc4p) is a long chain polyP kinase localized in the yeast vacuole. Here, we report that Vtc4 gene orthologs in Trypanosoma brucei (TbVtc4) and T. cruzi (TcVtc4) encode, in contrast, short chain polyP kinases that localize to acidocalcisomes. The subcellular localization was demonstrated by fluorescence and electron microscopy of cell lines expressing C-terminal tagged versions of these proteins. Recombinant TbVtc4 and TcVtc4 were expressed in bacteria, and polyP kinase activity was assayed in vitro. A reverse genetics approach allowed us to study the physiological role of TbVtc4 in bloodstream form (BSF) and procyclic form (PCF) trypanosomes. Our results indicate the enzyme is important for parasite survival in vitro and in vivo. We conclude that TbVtc4 is required for osmoregulation and virulence. Finally, we used biotinylated polyP to obtain the polyP-binding proteome of T. brucei PCF and T. cruzi epimastigotes. We identified 73 and 60 potential polyP-binding proteins in T. brucei and T. cruzi respectively, 36 of which were observed in both datasets. The most relevant proteins found these proteomes were: glycosomal malate dehydrogenase, hexokinase, glycosomal phosphoenolpyruvate carboxykinase, snoRNP protein GAR1, and ribosomal proteins S6, S8, L36 and L38. Additional experimental work would be necessary to validate this proteomic data. TbVtc4-KO mutant cell line generated in BSF would be a key tool to study the role of polyP in the activity of potential polyP-binding proteins found in T. brucei.