Unusually for a eukaryote,Trypanosoma bruceitranscribes its variant surface glycoprotein (VSG) gene expression sites (ESs) in a monoallelic fashion using RNA polymerase I (Pol I). It is still unclear how ES transcription is controlled inT. brucei.Here, we show that the TDP1 architectural chromatin protein is an essential high mobility group box (HMGB) protein facilitating Pol I transcription inT. brucei.TDP1 is specifically enriched at the active compared with silentVSGES and immediately downstream of ribosomal DNA promoters and is abundant in the nucleolus and the expression site body subnuclear compartments. Distribution of TDP1 at Pol I-transcribed loci is inversely correlated with histones. Depletion of TDP1 results in up to 40–90% reduction inVSGand rRNA transcripts and a concomitant increase in histones H3, H2A and H1 at these Pol I transcription units. TDP1 shares features with theSaccharomyces cerevisiaeHMGB protein Hmo1, but it is the first architectural chromatin protein facilitating Pol I-mediated transcription of both protein coding genes as well as rRNA. These results show that TDP1 has a mutually exclusive relationship with histones on actively transcribed Pol I transcription units, providing insight into how Pol I transcription is controlled.