Oil addition is challenging during high-moisture extrusion due to the negative fiber formation effects. A previous study found that oil-in-water (O/W) emulsions could significantly increase the oil content in high-moisture extrudates, but the molecular mechanism remained unclear. This study aimed to determine O/W emulsion influence on protein physicochemical properties in SPI extrudates during high-moisture extrusion. O/W emulsions were mixed with soy protein isolates (SPI) to prepare extrudates with oil/water ratios of 0/65, 4/61, and 8/57 (w/w). SDS-PAGE and ATR-FTIR analysis showed that higher oil/water ratios enhanced protein aggregation and promoted alteration from β-sheet to random coil in SPI extrudates, which could be correlated to the reduction of protein solubility. The color was altered to lighter and yellow, and hardness, chewiness, and fiber degree decreased with increased oil/water ratios in SPI extrudates. In addition, in vitro digestion analyses showed that higher oil content contributed to improved protein digestibility.