Updated project metadata. Force is transmitted between skeletal muscle and tendon via a highly specialized tissue interface: the myotendinous junction (MTJ). Despite its crucial role in mechano-transduction and involvement in muscle strain injury, knowledge about the molecular composition of the MTJ is incomplete. We performed LCMS-based proteomic analysis of human MTJ by progressive removal of the muscle component in the human semitendinosus muscle-tendon complex. Patellar tendon, which does not contains muscle tissue, was used as control. We quantified >3000 proteins across all samples and 112 proteins were significantly increased in MTJ. Of these, 24 proteins have previously been identified as MTJ proteins, including the MTJ-marker collagen XXII. Moreover, an overrepresentation of proteins related to sarcoglycan complex, laminin complex, cell junction, membrane, adhesion and extracellular matrix was observed among these proteins. We further confirmed the presence of newly identified MTJ proteins by immunofluorescence in human MTJ: Xin actin-binding repeat-containing protein 2 (XIRP2), thrombospondin-4 (THBS4), tetraspanin-24 (CD151), kindlin-2 (FERMT2), cartilage intermediate layer protein 1 (CILP) and integrin-alpha10 (ITGA10). Altogether, these data provide the first detailed proteomics resource of human MTJ.