Putative TTAGGG repeat-binding factor (TRF) homologues were identified in the genomes of Trypanosoma brucei, Trypanosoma cruzi and Leishmania major. They have significant sequence similarity to higher eukaryotic TRFs in their C-terminal DNA-binding myb domains, but only weak similarity in their N-terminal domains. T. brucei TRF is essential and was shown to bind to duplex TTAGGG repeats. RNAi-mediated knock-down of tbTRF arrested bloodstream cells at G2/M and procyclic cells partly at S phase. Functionally, tbTRF resembles mammalian TRF2 more than TRF1, as knock-down diminished telomere single-stranded G-overhang signals. This suggests that tbTRF, like vertebrate TRF2, is essential for telomere end protection, and supports the hypothesis that TRF rather than Rap1 is the more ancient DNA-binding component of the telomere protein complex. Identification of the first T. brucei telomere DNA binding protein and characterization of its function provides a new route to explore the roles of telomeres in pathogenesis of this organism. This work also establishes T. brucei as an attractive model for telomere biology.