The ADP ribosylation factors (Arfs) are a highly conserved subfamily of the Ras small GTPases with crucial roles in vesicle budding and membrane trafficking. Unlike in other eukaryotes, the orthologue of Arf1 in the host bloodstream form of Trypanosoma brucei is essential for the maintenance of endocytosis. In contrast, as shown in this study, knockdown of TbARF1 by RNA interference has no effect on fluid-phase endocytosis in the insect stage of the parasite. The protein remains essential for the viability of these procyclic cells but the major effect of TbARF1-depletion is enlargement of the lysosome. Our data indicate that protein trafficking and lysosomal function are differentially regulated by multiple factors, including TbARF1, during progression through the T. brucei lifecycle.