Phosphatidylethanolamine (PE) and phosphatidylserine (PS) are ubiquitously expressed and metabolically interconnected glycerophospholipids in eukaryotes and prokaryotes. In Trypanosoma brucei, PE synthesis has been shown to occur mainly via the Kennedy pathway, one of the three routes leading to PE synthesis in eukaryotes, while PS synthesis has not been studied experimentally. We now reveal the importance of T. brucei PS synthase 2 (TbPSS2) and T. brucei PS decarboxylase (TbPSD), two key enzymes involved in aminophospholipid synthesis, for trypanosome viability. By using tetracycline-inducible down-regulation of gene expression and in vivo and in vitro metabolic labeling, we found that TbPSS2 (i) is necessary for normal growth of procyclic trypanosomes, (ii) localizes to the endoplasmic reticulum and (iii) represents the unique route for PS formation in T. brucei. In addition, we identified TbPSD as type I PS decarboxylase in the mitochondrion and found that it is processed proteolytically at a WGSS cleavage site into a heterodimer. Down-regulation of TbPSD expression affected mitochondrial integrity in both procyclic and bloodstream form trypanosomes, decreased ATP production via oxidative phosphorylation in procyclic form and affected parasite growth.