Mechanisms of regulation of mitochondrial metabolism in trypanosomes are not completely understood. Here we present evidence that the Trypanosoma brucei mitochondrial calcium uniporter (TbMCU) is essential for the regulation of mitochondrial bioenergetics, autophagy and cell death, even in the bloodstream forms that are devoid of a functional respiratory chain and oxidative phosphorylation. Localization studies reveal its co-localization with MitoTracker staining. TbMCU overexpression increases mitochondrial Ca(2+) accumulation in intact and permeabilized trypanosomes, generates excessive mitochondrial reactive oxygen species (ROS) and sensitizes them to apoptotic stimuli. Ablation of TbMCU in RNAi or conditional knockout trypanosomes reduces mitochondrial Ca(2+) uptake without affecting their membrane potential, increases the AMP/ATP ratio, stimulates autophagosome formation and produces marked defects in growth in vitro and infectivity in mice, revealing its essentiality in these parasites. The requirement of TbMCU for proline and pyruvate metabolism in procyclic and bloodstream forms, respectively, reveals its role in regulation of mitochondrial bioenergetics.