Colicin M is an enzymatic bacteriocin produced by some Escherichia coli strains which provokes cell lysis of competitor strains by hydrolysis of the cell-wall peptidoglycan lipid II precursor. The overexpression of a gene, cbrA (yidS), was shown to protect E. coli cells from the deleterious effects of this colicin but the underlying resistance mechanism was not established. We here report that a major structural modification of the undecaprenyl-phosphate carrier lipid and of its derivatives occurred in membranes of CbrA-overexpressing cells, which explains the acquisition of resistance towards this bacteriocin. Indeed, a main fraction of these lipids, including the lipid II peptidoglycan precursor, now display a saturated isoprene unit at the α-position, i.e. the unit closest to the colicin M cleavage site. Only unsaturated forms of these lipids were normally detectable in wild-type cells. In vitro and in vivo assays showed that colicin M did not hydrolyze α-saturated lipid II, clearly identifying this substrate modification as the resistance mechanism. These saturated forms of undecaprenyl-phosphate and lipid II remained substrates of the different enzymes participating in peptidoglycan biosynthesis and carrier lipid recycling, allowing this colicin M-resistance mechanism to occur without affecting this essential pathway. IMPORTANCE Overexpression of the chromosomal cbrA gene allows E. coli to resist to colicin M (ColM), a bacteriocin specifically hydrolyzing the lipid II peptidoglycan precursor of targeted cells. This resistance results from a CbrA-dependent modification of the precursor structure, i.e. reduction of the α-isoprenyl bond of C55-carrier lipid moiety that is proximal to ColM cleavage site. This modification, observed here for the first time in eubacteria, annihilates the ColM activity without affecting peptidoglycan biogenesis. These data, which further increase our knowledge on the substrate specificity of this colicin, highlight the capability of E. coli to generate reduced forms of C55-carrier lipid and its derivatives. Whether the function of this modification is only relevant with respect to ColM resistance is now questioned.