[Background] Brochothrix thermosphacta is the main spoilage bacterium in Crucian Carp (Carassius auratus) during storage at 4 °C. Plantaricin 163, a new broad-spectrum bacteriocin produced by Lactobacillus plantarum, significantly extends the shelf life of crucian carp. [Objective] To investigate the antibacterial activity and mechanism of action of Plantaricin 163 against B. thermosphacta. [Methods] The minimum inhibitory concentration and bactericidal kinetics of Plantaricin 163 were determined to measure the antimicrobial effect of Plantaricin 163 on B. thermosphacta. Change in conductivity, and nucleic acid and protein leakage were measured, and flow cytometry, scanning electron microscopy, and transmission electron microscopy were performed, to determine the mechanism of action of Plantaricin 163 against B. thermosphacta. [Results] The MIC of Plantaricin 163 against B. thermosphacta was found to be 32 μg/mL, which was better than that of nisin, and the mode of action of the bacteriocin was killing of cells instead of growth inhibition. The permeability of the cell membrane was increased and caused a change in extracellular conductivity, thereby destroying the integrity of the cell membrane and resulting in the leakage of cell contents. The external morphology and internal structure of the cells were affected, eventually leading to cell death. [Conclusion] Plantaricin 163 can destroy the membrane and internal structure of B. thermosphacta.