Abstract Mechanisms of bacterial predation are crucial for revealing microbial adaptation strategies and interaction behaviors in the environment, yet they remain poorly understood. Previously, predators were reported to localize prey via specific cues. However, the process and mechanisms by which these cues, including signaling molecules, mediate predator localization remain unclear. Herein, we investigate the dynamic interaction between the predatory bacteria Lysobacter enzymogenes and its prey bacteria. By integrating genetic manipulation, transcriptomic analysis, biochemical assays, and live-cell tracking microscopy at the single-cell level, we present a novel predation strategy mediated by peptidoglycan hydrolase LssL, named peptidoglycan hydrolase-driven Prey Localization and Utilization System (phPLUS). In phPLUS, predators secrete LssL to initiate the Step I of the localization process. LssL then hydrolyzes prey and releases small molecules of glycine, which serve as signaling cues to guide the predator’s directional movement and promote the Step II of localization. In turn, prey signals upregulate the expression of LssL, which synergize with type VI secretion system to ultimately mediate prey killing through a novel regulatory pathway. This study reveals a new two-step localization strategy in bacterial predation, highlighting a previously unrecognized predation process and signal regulation mechanism, and expanding our understanding of predator–prey interactions and microbial ecological dynamics.