The effect of Ultra-High Pressure Homogenization (UHPH, 100–300MPa) on the physicochemical properties of oil-in-water emulsions prepared with 4.0% (w/v) of soy protein isolate (SPI) and soybean oil (10 and 20%, v/v) was studied and compared to emulsions treated by conventional homogenization (CH, 15MPa). CH emulsions were prepared with non-heated and heated (95°C for 15min) SPI dispersions. Emulsions were characterized by particle size determination with laser diffraction, rheological properties using a rotational rheometer by applying measurements of flow curve and by transmission electron microscopy. The variation on particle size and creaming was assessed by Turbiscan® analysis, and visual observation of the emulsions was also carried out. UHPH emulsions showed much smaller d3.2 values and greater physical stability than CH emulsions. The thermal treatment of SPI prior CH process did not improve physical stability properties. In addition, emulsions containing 20% of oil exhibited greater physical stability compared to emulsions containing 10% of oil. Particularly, UHPH emulsions treated at 100 and 200MPa with 20% of oil were the most stable due to low particle size values (d3.2 and Span), greater viscosity and partial protein denaturation. These results address the physical stability improvement of protein isolate-stabilized emulsions by using the emerging UHPH technology. •Emulsions were treated by conventional and Ultra-High Pressure Homogenization.•Physicochemical properties of emulsions were studied.•Emulsions treated at 100 and 200MPa with 20% of oil were the most stable.•Physical stability depended on the particle size, rheology, and protein denaturation.