A novel hyaluronidase (BgHya1) from Yak testis was isolated and shown to have compara-tively high activity on sodium hyaluronate. However, surveys on BgHya1 are still limited. The enzyme was purifiedthrough gel filtration on Sephacryl S-100 and cation-exchange on SP Sepharose fast flow; the purity was confirmedby a reverse phase FPLC Shodex C4 column. The specific activity of the purified BgHya1 was 20.4 U/mg assayed bythe colorimetric method against 0.85 U/mg for the crude enzyme, representing a 24-fold purification. It was a monomericprotein of 55 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and SephacrylS-200. It exhibited maximum activity in the presence of 0.15 M NaCl at 37 oC, pH 3.8, and a specificity to sodiumhyaluronate higher than that of chondroitin-4-sulfate, chondroitin-6-sulfate, and dermatan. The Km value for BgHya1,using sodium hyaluronate as substrate, was 0.106 mg/mL. Activity of BgHya1 was inhibited mildly by Ca2+and Fe2+,and significantly by Fe3+, Mg2+, EDTA, urea, heparin, and 0.5 M NaCl. It was not affected by Cu2+,Zn2+,Co2+, ascorbicacid, PMSF, DTT, glutathione (reduced), or L-cysteine. BgHya1 was shown to be heat unstable in the range of 4-45 oC. In terms of storage stability, 92% of the activity was retained after four weeks at 4 oC, and 58% at room temperature. In addition, adding BSA (1.0 mg/mL) to the enzyme sample prior to freezing resulted in complete retention of enzymeactivity. This work yielded a high purity hyaluronidase, the first one isolated from by-product.