Streptococcus pneumoniae hyaluronate lyase (spnHL) is a pathogenic bacterial spreading factor and cleaves hyaluronan, an important constituent of the extracellular matrix of connective tissues, through an enzymatic β-elimination process, different from the hyaluronan degradation by hydrolases in animals. The mechanism of hyaluronan binding and degradation was proposed based on the 1.56 Å resolution crystal structure, substrate modeling and mutagenesis studies on spnHL. Five mutants, R243V, N349A, H399A, Y408F and N580G, were constructed and their activities confirmed our mechanism hypothesis. The important roles of Tyr408, Asn349 and His399 in enzyme catalysis were proposed, explained and confirmed by mutant studies. The remaining weak enzymatic activity of the H399A mutant, the role of the free carboxylate group on the glucuronate residue, the enzymatic behavior on chondroitin and chondroitin sulfate, and the small activity increase in the N580G mutant were explained based on this mechanism. A possible function of the C-terminal β-sheet domain is to modulate enzyme activity through binding to calcium ions.