Abstract: To investigate the effect of high hydrostatic pressure on the enzymatic hydrolysis of high molecular weight hyaluronic acid (HMW-HA). This study adopted high hydrostatic pressure (0.1, 50, 100, 150 and 200 MPa) to assist bovine testicular hyaluronidase in hydrolyzing HMW-HA. Subsequently, the degree of hydrolysis and molecular weight of the hydrolysate were measured to investigate the effects of pressure, hydrolysis time, and enzyme concentration on the hydrolysis results, thereby determining optimal reaction conditions. Additionally, the chemical structure and thermal stability of the hydrolysis products were characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), nuclear magnetic resonance (NMR), and differential scanning calorimetry (DSC). The results indicate that bovine testicular hyaluronidase at a concentration of 40,000 U/g is suitable for the preparation of low molecular weight hyaluronic acid (LMW-HA) under pressure treatments ranging from 0.1, 50, 100, 150 and 200 MPa. Pressures of 50 MPa and 100 MPa effectively control the excessive hydrolysis of HMW-HA into HA oligosaccharides, increasing the LMW-HA content. The 100 MPa treatment yielded the highest LMW-HA content at 65.5%, representing a 19.9% increase compared to atmospheric pressure. However, treatment with excessively high pressure (150 MPa, 200 MPa) inhibits bovine testicular hyaluronidase from hydrolyzing HMW-HA. FT-IR and NMR results indicate that high hydrostatic pressure treatment does not significantly disrupt the chemical structure of hydrolysates. However, XRD analysis reveals that high pressure markedly disrupts their crystalline structure, with crystallinity decreasing as pressure increases. Additionally, DSC results indicate that lower pressure treatments (50 MPa, 100 MPa) have a minor effect on the thermal stability of hydrolysate products, whereas higher pressure treatments (150 MPa, 200 MPa) significantly (P<0.0.5) alter their thermal stability. In summary, high hydrostatic pressure treatment at appropriate pressure can inhibit the hydrolysis of HMW-HA into oligosaccharides while minimally affecting its chemical structure, thereby increasing the yield of low molecular weight hyaluronic acid.