Trypanosome brucei, the causative agent of African sleeping sickness, harbours a highly ordered, subpellicular microtubule cytoskeleton that defines many aspects of morphology, motility and virulence. This array of microtubules is associated with a large number of proteins involved in its regulation. Employing proximity-dependent biotinylation assay (BioID) using the well characterized cytoskeleton-associated protein CAP5.5 as a probe we identified CAP50 (Tb927.11.2610). This protein colocalizes with the subpellicular cytoskeleton microtubules but not with the flagellum. Depletion by RNAi results in defects in cytokinesis, morphology and partial disorganization of microtubule arrays. Published proteomics data indicate a possible association of CAP50 with two other, yet uncharacterized, cytoskeletal proteins, CAP52 (Tb927.6.5070) and CAP42 (Tb927.4.1300), which were therefore included in our analysis. We show that their depletion causes phenotypes similar to those described for CAP50 and that they are essential for cellular integrity.