A Disintegrin and Metalloproteinase with ThromboSpondin Motif (ADAMTS)-5 is considered to play a key role in degradation of aggrecan during the progression of osteoarthritis (OA). Thus, ADAMTS-5 inhibitors are emerging as potential disease-modifying OA drugs. The generation of small molecule inhibitors of metalloproteinases has not achieved successful outcomes primarily due to the lack of selectivity. On the other hand, monoclonal antibodies were proved potent and selective binders of many biologically relevant targets. We therefore aimed to isolate inhibitory antibodies against ADAMTS-5. A human naïve single-chain variable fragment library was used to select and isolate anti-ADAMTS-5 antibodies. Among the isolated antibodies, clones 2D3 and 2D5 inhibited ADAMTS-5 cleavage both of a peptide substrate (Ki app values of 2 nM and 18 nM, respectively) and bovine aggrecan (Ki app values of 30 nM and 300 nM, respectively). Antibody 2B9 did not inhibit ADAMTS-5 activity against the peptide substrate, but it inhibited the cleavage of aggrecan (Ki app: 300 nM). Surface plasmon resonance studies with a series of deletion mutants of ADAMTS-5 indicated that 2D3 and 2D5 bind to the catalytic/disintegrin domain (KD values of 3 nM and 55 nM, respectively), whereas 2B9 binds to the spacer domain of this enzyme (KD value of 6 nM), thus being an exosite inhibitor. Kinetic studies indicated that the anti-ADAMTS-5 antibodies described here do not act through a zinc-chelating mechanism of inhibition. Moreover, inhibition and co-immunoprecipitation data suggested that for some antibodies, such as 2D5, the binding site is different from that of TIMP-3. The efficacy of ADAMTS-5-blocking antibodies was investigated using model systems of aggrecan degradation (IL-1α/oncostatin M-stimulated porcine and human articular cartilage explants). The results indicated that antibodies 2D3 and 2B9 effectively inhibited aggrecanase activity in these systems. These antibodies may therefore be useful as chondroprotectant agents in OA.