It has been demonstrated that Mycobacterium tuberculosis can utilize hyaluronan (HA) as an alternative carbon resource; however, the gene responsible for HA utilization has not been identified. We overexpressed the soluble product of the Rv0394c gene from M. tuberculosis H37Rv in Escherichia coli and purified it using affinity chromatography and anion exchange chromatography. The hyaluronidase and chondrosulfatase activities of the purified recombinant protein Rv0394c (rRv0394c) were detected and quantitatively measured. Analysis of nucleotide and derived amino acid sequences of the Rv0394c gene revealed that homologs of this gene were conserved in pathogenic mycobacteria, but absent in non-pathogenic mycobacteria. In the current study, we provide novel identification and characterization of a gene encoding hyaluronidase and chondrosulfatase in M. tuberculosis.