Bacteriocins are ribosomally synthesized antimicrobial peptides or proteins produced by bacteria, which usually inhibit the growth of closely related species. Bacteriocins produced by Bacillus species have attracted emerging interests by both food and pharmaceutical industries due to their broader antimicrobial spectrum, compared with the widely used bacteriocins produced by lactic acid bacteria. Thurincin H is an anti-listerial bacteriocin produced by Bacillus thuringiensis SF361, a strain originally isolated from US domestic honey. Purified thurincin H exhibited a broad antimicrobial activity against various foodborne pathogens and spoilage microorganisms across several genera, including Bacillus, Carnobacterium, Geobacillus, Enterococcus, Listeria, and Staphylococcus. A rapid and simple method was developed to produce and purify large amounts of thurincin H. The purified thurincin H was characterized regarding its thermal and acid stability, and its inhibitory effect against B. cereus spores. Systematic conservative and non-conservative site directed mutagenesis were performed to identify the critical amino acids in the native thurincin H production pathway, using an optimized thurincin H heterologous expression system newly developed in this study. The thurincin H gene cluster was confirmed by heterologously expressing a bioinformatically identified gene cluster in a sensitive B. thuringiensis strain. The sensitive strain acquired complete immunity and produced thurincin H at a higher level compared with the natural producer. The bactericidal thurincin H caused cell morphology changes in a concentration dependent manner, but did not induce cell membrane permeability, which indicated a novel mode of action that is different from the generalized pore-forming mechanism for most bacteriocins.