Trypanosoma brucei, the causative agent of African Sleeping sickness, is replete with unique biochemistry, including unusual features of gene transcription. The parasite also contains over 4500 non-annotated genes, representing novel biochemistry yet to be explored. Using tandem affinity purification (TAP)-tagged TbTFIIB, we identified and subsequently confirmed, one of the non-annotated Trypanosoma brucei proteins, Tb11.02.4300, as a TbTFIIB-interacting protein. The 49 kDa protein is nuclear and essential for parasite variability as determined by RNA interference studies; hence, the nomenclature T. brucei Essential Nuclear Factor (TbENF). TbENF is shown to interact with DNA in a sequence-independent fashion under the conditions examined. Furthermore, TbENF bears motifs associated with many eukaryotic transcription factors, such as a glutamine-rich region and a leucine zipper, yet TbENF is specific to trypanosomatids making it a potentially attractive therapeutic target. Taken together, our results suggest a role for TbENF in trypanosome gene transcription.