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 Tb BILBO1. In searching for novel immune-tools to study this protein, we raised nanobodies against Tb BILBO1. Nanobodies (Nb) that were selected according to their binding properties to Tb BILBO1, were 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 Tb BILBO1 RNAi knockdown. Our results validate the feasibility of generating functional single-domain antibody derived intrabodies to target trypanosome cytoskeleton proteins.