Soybean protein isolate (SPI) adsorbed on the oil-water interface was used as an emulsifier and stabilizer to form an oil-in-water (O/W) emulsion (2.0 wt% protein and 20.0 wt% soybean oil). The characteristics of SPI-stabilized emulsions, including changes in particle size, zeta potential, and microstructure, under the action of simulated gastric fluid (37 ℃, pH 1.2, and 34 mM NaCl ionic strength, with continuous shaking at approximately 95 r/min for 2 h) were investigated. The microstructure and diameter of the emulsion showed that SPI (pH 7.0) was capable of forming a stable emulsion, but underwent extensive droplet flocculation or coalescence when mixed with simulated gastric fluid. The degree of droplet flocculation or coalescence markedly depended upon hydrolysis time. The zeta-potential values gradually changed from -35.9±0.3 mV to +16.3±0.9 mV after the emulsion was mixed with simulated gastric fluid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that when SPI was present at the interface of the O/W emulsion, it was more easily hydrolyzed by pepsin as compared to native SPI solution. This study improves our understanding of the characteristics of an O/W emulsion stabilized by SPI in simulated gastric fluid and has high significance for guiding the design of food systems based on SPI emulsions.