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Since the 1970s, numerous reports have described elevated hexosaminidase activities in rheumatoid arthritis. However, due to the overlapping substrate specificities of different hexosaminidases, identification of the exact enzyme(s) responsible for the elevated activity remains incomplete. In this work we tested if the recently described enzyme, hexosaminidase D was expressed in human arthritic joints, and could contribute to the elevated hexosaminidase activity in rheumatoid arthritis. Thermostable β-d-N-acetyl-galactosaminidase (hexosaminidase D) activities were determined in synovial fluid samples, synovial membranes, synovial fibroblast cell strains and synovial fibroblast-derived extracellular vesicles of patients with rheumatoid arthritis and osteoarthritis using chromogenic substrates. Expression of the HEXDC gene was detected both in steady state and in TGF-β treated synovial fibroblasts by real time PCR. Strikingly, hexosaminidase D accounted for approximately 50% of the total β-N-acetyl-galactosaminidase activity in synovial membranes and synovial fibroblasts, and it was responsible for the vast majority of the β-d-N-acetyl-galactosaminidase activity in synovial fluid samples. TGF-β downregulated the expression of hexosaminidase D in synovial fibroblasts dose-dependently. Of note, significant activity of hexosaminidase D was also found in association with extracellular vesicles released by synovial fibroblasts. This first study that describes the expression and disease relevance of the HEXDC gene in humans demonstrates the expression of this novel enzyme within the joints, and suggests that its activity may significantly contribute to the overall local exoglycosidase activity.