Updated project metadata. Trypanosoma brucei spp. develop into mammalian-infectious metacyclic trypomastigotes inside tsetse salivary glands. Besides acquiring a variant surface glycoprotein (VSG) coat, little is known about the metacyclic expression of invariant surface antigens. Proteomics analyses of saliva from T. brucei-infected flies identified, in addition to VSG and Brucei Alanine-Rich Protein (BARP) peptides, a family of GPIanchored surface proteins herein named Metacyclic Invariant Surface Proteins (MISP). The MISP family is encoded by five paralog genes with >80% protein identity, which are exclusively expressed by salivary gland stages of the parasite and peak in metacyclic stage, as shown by confocal microscopy and immuno-high resolution scanning electron microscopy. Crystallographic analysis of a MISP isoform (MISP360) and a high confidence model of BARP revealed a triple helical bundle architecture commonly found in other trypanosome surface proteins. Molecular modelling combined with live fluorescent microscopy suggests that MISP N-termini are extended above the VSG coat. However, vaccination with recombinant MISP360 isoform did not protect mice against a T. brucei infectious tsetse bite. Lastly, both RNAi knock down and CRISPR-Cas9-driven knock out of all MISP paralogues suggest they are not essential for parasite development in the tsetse vector. We speculate that MISP may be relevant during trypanosome inoculation or establishment in the vertebrate’s skin.