Trypanosoma brucei belongs to a genus of protists that cause life-threatening and economically important diseases of human and animal populations in Sub-Saharan Africa. T. brucei cells are covered in surface glycoproteins, some of which are used to escape the host immune system. Exo-/endocytotic trafficking of these and other molecules occurs via a single copy organelle called the flagellar pocket (FP). The FP is maintained and enclosed around the flagellum by the flagellar pocket collar (FPC). To date, the most important cytoskeletal component of the FPC is an essential calcium-binding, polymer-forming protein called TbBILBO1. In searching for novel tools to study this protein, we raised nanobodies (Nb) against purified, full-length TbBILBO1. Nanobodies were selected according to their binding properties to TbBILBO1, tested as immunofluorescence tools, and expressed as intrabodies (INb). One of them, Nb48, proved to be the most robust nanobody and intrabody. We further demonstrate that inducible, cytoplasmic expression of INb48 was lethal to these parasites, producing abnormal phenotypes resembling those of TbBILBO1 RNA interference (RNAi) knockdown. Our results validate the feasibility of generating functional single-domain antibody-derived intrabodies to target trypanosome cytoskeleton proteins. IMPORTANCE Trypanosoma brucei belongs to a group of important zoonotic parasites. We investigated how these organisms develop their cytoskeleton (the internal skeleton that controls cell shape) and focused on an essential protein (BILBO1) first described in T. brucei. To develop our analysis, we used purified BILBO1 protein to immunize an alpaca to make nanobodies (Nb). Nanobodies are derived from the antigen-binding portion of a novel antibody type found only in the camel and shark families of animals. Anti-BILBO1 nanobodies were obtained, and their encoding genes were inducibly expressed within the cytoplasm of T. brucei as intrabodies (INb). Importantly, INb48 expression rapidly killed parasites producing phenotypes normally observed after RNA knockdown, providing clear proof of principle. The importance of this study is derived from this novel approach, which can be used to study neglected and emerging pathogens as well as new model organisms, especially those that do not have the RNAi system.Read less <