Updated publication reference for PubMed record(s): 27744090. Actin and tubulin are rather conserved and easy to trace throughout eukaryotic evolution, but less so proteins of the intermediate filament (IF) family. Canonical IF protein families appear restricted to Metazoa, although protists are well known for an extensive number of filamentous structures that neither involve actin nor tubulin, yet support the complex cell architecture. Trichomonadida are an evolutionary early branching group of excavate parasites that are united by a unique cytoskeletal framework, which includes the costa. This rod-like structure supports the flagellum that is attached to the undulating membrane. We determined the proteome of the detergent resistant cytoskeletal backbone of Tetratrichomonas gallinarum (the pelta axostyle costa karyomastigont complex, PACK) to identify the proteins that constitute these structures and to investigate their properties. Among the 582 proteins identified in each of the three individual proteome screens, we found major components of the costa whose association was verified through in vivo localization experiments. Intriguingly, many of the PACK proteins are characterized by recognizable features, including long central coiled-coil regions, that are reminiscent of metazoan IF proteins. The newly identified cytoskeletal proteins cannot be attributed to any known proteins of the metazoan IF families, albeit they resemble these in terms of their predicted tertiary structure and the more general function. Our data offer further support that IF proteins of protists, which form cytoskeletal filaments that do not depend on actin and tubulin, evolved many times independently in eukaryotes.