Phospholipase A1 (PlaA) plays a pivotal role in diverse applications within the food and biochemical medical industries. Herein, we investigate the impact of the accessory protein encoded by plaS from Serratia marcescens on PlaA activity in Escherichia coli. Notably, PlaS demonstrates the ability to enhance PlaA activity while concurrently exhibiting inhibitory effects on the growth of E. coli BL21 (DE3). Our study revolves around probing the inhibitory action of PlaS on E. coli BL21 (DE3). PlaS exhibits a propensity to heighten both the permeability of outer and inner cell membranes, leading to concomitant reductions in membrane fluidity and surface hydrophobicity. This phenomenon is validated through scanning electron microscopy (SEM) analysis, which highlights PlaS's capacity to compromise membrane integrity. Moreover, through a comprehensive comparative transcriptomic sequencing approach, we identify four down-regulated genes (galM, ybhC, ldtC, and kdpB) alongside two up-regulated genes (rbsB and degP). These genes are intricately associated with processes such as cell membrane synthesis and modification, energy metabolism, and transmembrane transport. Our investigation unveils the intricate gene-level mechanisms underpinning PlaS-mediated growth inhibition and membrane disruption. Consequently, our findings serve as a significant reference for the elucidation of membrane protein mechanisms, shedding light on potential avenues for future exploration.