Hyaluronidases (Hyals) are broadly used in medical applications to facilitate the dispersion and/or absorption of fluids or medications. This study reports the isolation, cloning, and industrial-scale recombinant production, purification and full characterization, including X-ray structure determination at 1.45 A, of an extracellular Hyal from the nonpathogenic bacterium Streptomyces koganeiensis. The recombinant S. koganeiensis Hyal (rHyal_Sk) has a novel bacterial catalytic domain with high enzymatic activity, compared with commercially available Hyals, and is more thermostable and presents higher proteolytic resistance, with activity over a broad pH range. Moreover, rHyal_Sk exhibits remarkable substrate specificity for hyaluronic acid (HA) and poses no risk of animal cross-infection.