KU is a heterodimer, consisting of [~]70 and [~]80 kDa subunits (KU70 and KU80, respectively), which is involved in a variety of nuclear functions. We generated tbKU80-deficient trypanosomes to explore the potential role of the tbKU complex in telomere maintenance and transcriptional regulation of variant surface glycoprotein (VSG) genes in Trypanosoma brucei. Using real-time PCR, we demonstrated that the expression of several different VSG genes remains tightly regulated in tbKU80-deficient bloodstream-form cell lines, suggesting that VSG transcription profiles do not change in these cells. Owing to developmental silencing of the VSG Expression Sites (ES), no VSG is transcribed in the insect procyclic stage. With a green fluorescent protein reporter system, we showed that tbKU80-deficient mutants are fully capable of ES silencing after differentiation into procyclic forms. Using T7 RNA polymerase to explore the transcriptional accessibility of ES chromatin in vivo, we demonstrated that tbKU80-deficient bloodstream-form cells were able to generate transcriptionally repressed ES chromatin after differentiation into procyclic cells. Finally, we demonstrated progressive telomere shortening in tbKU80-deficient mutants. The possible function of tbKU80 in telomere maintenance and regulation of telomerase is discussed.