The cyclic antimicrobial lipopeptide daptomycin triggers the LiaFSR membrane stress response pathway in enterococci and many other Gram-positive organisms. LiaR is the response regulator that, upon phosphorylation, binds in a sequence specific manner to DNA to regulate transcription in response to membrane stress. In clinical settings, nonsusceptibility to daptomycin by Enterococcus faecium is correlated frequently with a mutation in LiaR of Trp73 to Cys (LiaRW73C). We have determined the structure of the activated Enterococcus faecium LiaR protein at 3.2 Å resolution and, in combination with solution studies, show that activation of LiaR induces formation of a LiaR dimer that increases LiaR affinity at least 40-fold for the extended regulatory regions upstream of the liaFSR and liaXYZ operons. In vitro, LiaRW73C induces phosphorylation-independent dimerization of LiaR and provides a biochemical basis for non-susceptibility to daptomycin by upregulation of the LiaFSR regulon. A comparison of the Enterococcus faecalis LiaR, Enterococcus faecium LiaR and the LiaR homolog from Staphylococcus aureus (VraR) and the mutations associated with daptomycin resistance suggest that physicochemical properties such as oligomerization state and DNA specificity, though tuned to the biology of each organism, share some features that could be targeted for new antimicrobials.