A novel hyaluronidase (BgHya1) from Yak Bos grunniens testis was isolated and shown to have comparatively high activity on sodium hyaluronate. However, surveys on BgHya1 are still limited. The enzyme was purified through gel filtration on Sephacryl S-100 and cation-exchange on SP Sepharose fast flow; the purity was confirmed by a reverse phase FPLC Shodex C4 column. The specific activity of the purified BgHya1 was 20.4 U/mg assayed by the colorimetric method against 0.85 U/mg for the crude enzyme, representing a 24-fold purification. It was a monomeric protein of 55 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Sephacryl S-200. It exhibited maximum activity in the presence of 0.15 M NaCl at 37 ℃, pH 3.8, and a specificity to sodium hyaluronate 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+, ascorbic acid, PMSF, DTT, glutathione (reduced), or L-cysteine. BgHya1 was shown to be heat unstable in the range of 4-45 ℃. In terms of storage stability, 92% of the activity was retained after four weeks at 4 ℃, 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 enzyme activity. This work yielded a high purity hyaluronidase, the first one isolated from Bos grunniens by-product.