ABSTRACT Lactococcin 972 (Lcn972) is a cell wall-active bacteriocin that inhibits cell wall biosynthesis in Lactococcus lactis . In this work, the transcriptomes of the Lcn972-resistant (Lcn r ) mutant L. lactis D1 and its parent strain were compared to identify factors involved in Lcn972 resistance. Upregulated genes included members of the cell envelope stress (CesSR) regulon, the penicillin-binding protein pbpX gene and gene llmg2447 , which may encode a putative extracytoplasmic function (ECF) anti-sigma factor. The gene llmg2447 is located downstream of the nonfunctional ECF gene sigX pseudo . Nisin-controlled expression of llmg2447 led to high Lcn972 resistance in L. lactis , with no cross-resistance to other cell wall-active antimicrobials. Upregulation of llmg2447 in L. lactis D1 (Lcn r ) was linked to the integration of insertion element IS 981 into the llmg2447 promoter region, replacing the native −35 box and activating the otherwise silent promoter P 2447 . This is the first example of an orphan ECF anti-sigma factor involved in bacteriocin resistance. This new role in neutralizing cell wall-active compounds (e.g., Lcn972) could have evolved from a putative primary function of Llmg2447 in sensing cell envelope stress.