Aims To understand the bactericidal action of enterocin LD3 against Gram-negative bacteria. Methods and Results Minimum inhibitory concentration (MIC) of enterocin LD3 against Micrococcus luteus MTCC 106 and Escherichia coli NCDC 135 was 80 and 112 mu g ml(-1), and minimum bactericidal concentration (MBC) was 128 and 180 mu g ml(-1), respectively. The efflux of potassium ion (K+) was 14 and 13 ppm and electrical conductivity 10 center dot 5 and 8 center dot 3 mS cm(-1) in cell-free supernatant of MIC-treated cells of M. luteus and E. coli respectively. The increased absorbance (OD260/280) 0 center dot 422/0 center dot 260 and 0 center dot 110/0 center dot 075 in the bacteriocin-treated cells of M. luteus MTCC 106 and E. coli, NCDC 135, respectively, suggested the release of nucleic acids and proteins. The higher infrared absorbance at 1451 center dot 82 and similar to 1094 center dot 30 cm(-1) further suggested its interaction with cell membrane and nucleic acids of the target bacteria. The interaction of bacteriocin with nucleic acids was also confirmed using gel retardation assay. Transmission electron microscopy of the bacteriocin-treated cells revealed disruption of cell membrane and leakage of cytoplasmic contents. Conclusions Enterocin LD3 demonstrates bactericidal activity against Gram-negative bacteria interacting with cell membrane and nucleic acids. Significance and Impact of the Study The study discloses the possible mechanism of action of enterocin LD3 against Gram-negative bacteria which is a rare phenomenon.