Proteoglycan 4 (PRG4) is an extracellular matrix protein that maintains homeostasis through its boundary lubricating and anti-inflammatory properties. Altered expression and function of PRG4 have been associated with joint inflammatory diseases, including osteoarthritis (OA). We found that mast cell tryptase cleaves PRG4 in a dose- and time-dependent manner, which was confirmed by silver stain gel electrophoresis and mass spectrometry. Tryptase-treated PRG4 resulted in a reduction of lubrication. Compared to full-length, processed PRG4 was shown to further activate NF-B expression in cells overexpressing TLR2, -4, and -5. In the destabilization of the medial meniscus (DMM) model of OA in rat, tryptase and PRG4 colocalized at the site of injury in knee cartilage and were associated with disease severity. Primary synovial fibroblasts from OA patients or healthy subjects treated with tryptase and/or PRG4, and synovial cells from OA patients, were subjected to a quantitative shotgun proteomics and proteome changes were characterized, further supporting the role of NF-B activation. We identified tryptase as a modulator of joint lubrication via the processing of PRG4 in OA.