The bacterial cell membrane is an interface for cell envelope synthesis, protein secretion, virulence factor assembly, and a target for host cationic antimicrobial peptides (CAMPs). To resist CAMP killing, several Gram-positive pathogens encode the multiple peptide resistance factor (MprF) enzyme that covalently attaches cationic amino acids to anionic phospholipids in the cell membrane. While E. faecalis encodes twomprFparalogs, MprF2 plays a dominant role in conferring resistance to killing by the CAMP human β-defensin 2 (hBD-2) in E. faecalis strain OG1RF. The goal of the current study is to understand the broader lipidomic and functional roles of E. faecalismprF.We analyzed the lipid profiles of parental wild-type andmprFmutant strains and show that while ΔmprF2and ΔmprF1ΔmprF2mutants completely lacked cationic lysyl-phosphatidylglycerol (L-PG), the ΔmprF1mutant synthesized ~70% of L-PG compared to the parent. Unexpectedly, we also observed a significant reduction of PG in ΔmprF2and ΔmprF1ΔmprF2. In themprFmutants, particularly ΔmprF1ΔmprF2, the decrease in L-PG and phosphatidylglycerol (PG) is compensated by an increase in a phosphorus-containing lipid, glycerophospho-diglucosyl-diacylglycerol (GPDGDAG), and D-ala-GPDGDAG. These changes were accompanied by a downregulation ofde novofatty acid biosynthesis and an accumulation of long-chain acyl-acyl carrier proteins (long-chain acyl-ACPs), suggesting that the suppression of fatty acid biosynthesis was mediated by the transcriptional repressor FabT. Growth in chemically defined media lacking fatty acids revealed severe growth defects in the ΔmprF1ΔmprF2mutant strain, but not the single mutants, which was partially rescued through supplementation with palmitic and stearic acids. Changes in lipid homeostasis correlated with lower membrane fluidity, impaired protein secretion, and increased biofilm formation in both ΔmprF2and ΔmprF1ΔmprF2, compared to the wild type and ΔmprF1. Collectively, our findings reveal a previously unappreciated role formprFin global lipid regulation and cellular physiology, which could facilitate the development of novel therapeutics targeting MprF.IMPORTANCEThe cell membrane plays a pivotal role in protecting bacteria against external threats, such as antibiotics. Cationic phospholipids such as lysyl-phosphatidyglycerol (L-PG) resist the action of cationic antimicrobial peptides through electrostatic repulsion. Here we demonstrate that L-PG depletion has several unexpected consequences in Enterococcus faecalis, including a reduction of phosphatidylglycerol (PG), enrichment of a phosphorus-containing lipid, reduced fatty acid synthesis accompanied by an accumulation of long-chain acyl-acyl carrier proteins (long chain acyl-ACPs), lower membrane fluidity, and impaired secretion. These changes are not deleterious to the organism as long as exogenous fatty acids are available for uptake from the culture medium. Our findings suggest an adaptive mechanism involving compensatory changes across the entire lipidome upon removal of a single phospholipid modification. Such adaptations must be considered when devising antimicrobial strategies that target membrane lipids.