Aggrecanases that include ADAMTS1, 4, 5, 8, 9 and 15 are considered to play key roles in aggrecan degradation in osteoarthritic cartilage. Here we demonstrate that calcium pentosan polysulfate (CaPPS) directly inhibits the aggrecanase activity of ADAMTS4 without affecting the mRNA expression of the ADAMTS species in interleukin-1alpha-stimulated osteoarthritic chondrocytes. Synthetic peptides corresponding to specific regions of the thrombospondin type 1 repeat, cysteine-rich or spacer domain of ADAMTS4 inhibit the binding to immobilized CaPPS. These data suggest that CaPPS could function as chondroprotective agent for the treatment of osteoarthritis by inhibition of ADAMTS4 through interaction with the C-terminal ancillary domain.