Trypanosomatids are a large family of unicellular eukaryotes, many of which are parasites in higher eukaryotes including man. Much of our understanding of metabolism in these organisms has been gained form the study of the human infective representatives (Trypanosoma brucei subpecies, Trypanosoma cruzi and Leishmania spp.) which are transmitted by blood-feeding arthropods. The insect vectors of these parasites use proline as a principal carbon and energy source circulating in their haemolymph. Accordingly the insect-forms of the human infectious parasites have evolved to exploit abundant proline when in this environment, but being able to activate different biochemical pathways when in other environments. Interestingly, if glucose is available, metabolic capability can shift to make this carbohydrate the preferred substrate. Proline has also been shown to play key roles in osmoregulation, differentiation in representatives of the group and may even play a role in immunosuppression elicited by the American trypanosome T. cruzi. This review focuses on recent progress in understanding the different aspects of proline metabolism in trypanosomatids, with a particular interest on the insect forms.