Trypanosoma brucei is a protist parasite that infects human and animals, currently found in sub-Saharan Africa. T. brucei human infection is called African trypanosomiasis or sleeping sickness and it leads to death if untreated. The cattle disease (Nagana) has harsh socioeconomic impacts in developing nations affected. Inorganic phosphate (Pi) is vital for all organisms, as cells need it for the synthesis of essential compounds such as nucleotides and phospholipids. Deprivation or excess of Pi can be harmful, therefore normal metabolism requires a balance in cytosolic phosphate. One form of regulating Pi levels is to store it as polyphosphate (polyP), a polymer of phosphates that can be degraded to release Pi to the cytosol. The main organelles for polyP storage in T. brucei are the acidocalcisomes, acidic compartments that store phosphate, polyphosphate, calcium and other metal ions. The physiological regulation of acidocalcisomes is not well understood. This work demonstrated that signaling molecules named inositol phosphates are key regulators of acidocalcisome phosphate storage. We first studied the inositol pyrophosphate synthetic pathway of T. brucei and characterized the activity of inositol phosphate kinases TbIPMK, TbIP5K and TbIP6K. Our experiments revealed that T. brucei produces inositol hexakisphosphate (IP6) and 5-diphospho-inositol pentakisphosphate (5-IP7). We discovered that depletion of TbIPMK causes a reduction in polyphosphate synthesis in acidocalcisomes. Interestingly, we also observed that the sodium/phosphate symporter TbPho91, located in acidocalcisomes, is stimulated by 5-IP7. Additionally, TbPho91 knockout cells are more sensitive to DNA damage, revealing that an imbalance in cytosolic Pi may reduce nucleotide synthesis and consequently delay DNA repair. This mechanism for regulation of polyP synthesis in acidocalcisomes and phosphate release by the transporter Pho91 is also found in Saccharomyces cerevisiae, revealing a conserved mechanism of phosphate homeostasis regulation. We also investigated enzymes involved in control of polyP outside acidocalcisomes. Our research led to the identification and characterization of the polyphosphatase activity of two nudix hydrolases TbNH2 and TbNH4. TbNH2 was detected in glycosomes and TbNH4 in cytosol of the parasites. Current results suggest that nudix hydrolases may help control polyP levels in glycosomes and cytosol.