Chondroitinase B from Pedobacter heparinus is the only known enzyme strictly specific for dermatan sulfate and is a widely used enzymatic tool for the structural characterization of glycosaminoglycans. This beta-helical polysaccharide lyase belongs to family PL-6 and cleaves the beta(1,4) linkage of dermatan sulfate in a random manner, yielding 4,5-unsaturated dermatan sulfate disaccharides as the product. The previously reported structure of its complex with a dermatan sulfate disaccharide product identified the -1 and -2 subsites of the catalytic groove. We present here the structure of chondroitinase B complexed with several dermatan sulfate and chondroitin sulfate oligosaccharides. In particular, the soaking of chondroitinase B crystals with a dermatan sulfate hexasaccharide results in a complex with two dermatan sulfate disaccharide reaction products, enabling the identification of the +2 and +1 subsites. Unexpectedly, this structure revealed the presence of a calcium ion coordinated by sequence-conserved acidic residues and by the carboxyl group of the l-iduronic acid at the +1 subsite. Kinetic and site-directed mutagenesis experiments have subsequently demonstrated that chondroitinase B absolutely requires calcium for its activity, indicating that the protein-Ca(2+)-oligosaccharide complex is functionally relevant. Modeling of an intact tetrasaccharide in the active site of chondroitinase B provided a better understanding of substrate specificity and the role of Ca(2+) in enzymatic activity. Given these results, we propose that the Ca(2+) ion neutralizes the carboxyl moiety of the l-iduronic acid at the cleavage site, whereas the conserved residues Lys-250 and Arg-271 act as Brønsted base and acid, respectively, in the lytic degradation of dermatan sulfate by chondroitinase B.