Yeast contamination is a key factor affecting juice quality, and its control is of utmost importance. This study investigated the inactivation mechanism of Saccharomyces cerevisiae by combining medium-temperature-assisted high-voltage pulsed electric fields with vanillin (PVT) and its effect on red raspberry juice quality. The results showed that under the artificial neural network-genetic algorithm (ANN-GA) optimized conditions (39.34 kV/cm, 1.52 mg/mL vanillin, 60 s, 60 °C), PVT treatment inactivated Saccharomyces cerevisiae by 2.24 log CFU/mL. Mechanistic studies indicated that PVT treatment disrupted the permeability and fluidity of Saccharomyces cerevisiae cell membranes, leading to leakage of intracellular proteins and nucleic acids, reduced ATP content, and damage to the cell wall and membrane, ultimately causing cell deformation, rupture, and death. FT-IR analysis confirmed the changes in cellular structure. In contrast to conventional thermal treatment, PVT better preserved key quality attributes of raspberry juice, including vitamin C retention (>90 %), total phenolic content, and antioxidant capacity. This study provides an effective solution to ensure the microbial safety of juice while maintaining its quality to the greatest extent.