Bacteriocins, naturally derived antimicrobial peptides, are considered promising alternatives to traditional preservatives and antibiotics, particularly in food and medical applications. Despite extensive research on various bacteriocins, cyclic varieties remain understudied. This study introduces Gassericin GA-3.1, a novel cyclic bacteriocin produced by Lactobacillus gasseri LG145. We employed a multi-step purification process, including salt precipitation, ion-exchange chromatography, gel filtration chromatography, and ultimately high-performance liquid chromatography (HPLC), achieving a specific activity of 4660.89 AU/mg for the purified Gassericin GA-3.1. Mass spectrometry revealed a molecular mass of 5613.842 Da. Genome analysis confirmed Gassericin GA-3.1 as a novel class IIc bacteriocin with a unique amino acid sequence. Secondary structure prediction suggested the presence of three α-helices, two β-pleated strands, and a random coil. Physicochemical characterization demonstrated GassericinGA-3.1's thermal stability, resistance to pH extremes, surfactants, and broad-spectrum antibacterial potency. Notably, Gassericin GA-3.1 effectively inhibit Listeria monocytogenes through mechanism involving surface perforation, membrane potential disruption, and downregulation of virulence, biofilm formation, and motility genes. Overall, our finding position Gassericin GA-3.1 as a potential candidate for antimicrobial applications in the food and health industries.