The Arf-like (Arl) small GTPases have a diverse range of functions in the eukaryotic cell. Metazoan Arl2 acts as a regulator of microtubule biogenesis, binding to the tubulin-specific chaperone cofactor D. Arl2 also has a mitochondrial function through its interactions with BART and ANT-1, the only member of the Ras superfamily to be found in this organelle to date. In the present study, we describe characterization of the Arl2 orthologue in the protozoan parasite Trypanosoma brucei. Modulation of TbARL2 expression in bloodstream form parasites by RNA interference (RNAi) causes inhibition of cleavage furrow formation, resulting in a severe defect in cytokinesis and the accumulation of multinucleated cells. RNAi of TbARL2 also results in loss of acetylated alpha-tubulin but not of total -tubulin from cellular microtubules. While overexpression of TbARL2(myc) also leads to a defect in cytokinesis, an excess of untagged protein has no effect on cell division, demonstrating the importance of the extreme C-terminus in correct function. TbARL2 overexpressing cells (either myc-tagged or untagged) have an increase in acetylated -tubulin. Our data indicate that Arl2 has a fundamentally conserved role in trypanosome microtubule biogenesis that correlates with -tubulin acetylation.