The dynamic lipid composition of bacterial cytoplasmic membranes has a profound impact on vital bacterial fitness and susceptibility to membrane-damaging agents, temperature, or osmotic stress. However, it has remained largely unknown how changes in lipid patterns affect the abundance and expression of membrane proteins. Using recently developed gel-free proteomics technology, we explored the membrane proteome of the important human pathogen Staphylococcus aureus in the presence or absence of the cationic phospholipid lysyl-phosphatidylglycerol (Lys-PG). We were able to detect almost half of all theoretical integral membrane proteins and could reliably quantify more than 35% of them. It is worth noting that the deletion of the Lys-PG synthase MprF did not lead to a massive alteration but a very distinct up- or down-regulation of only 1.5 or 3.5% of the quantified proteins. Lys-PG deficiency had no major impact on the abundance of lipid-biosynthetic enzymes but significantly affected the amounts of the cell envelope stress-sensing regulatory proteins such as SaeS and MsrR, and of the SaeS-regulated proteins Sbi, Efb, and SaeP. These data indicate very critical interactions of membrane-sensory proteins with phospholipids and they demonstrate the power of membrane proteomics for the characterization of bacterial physiology and pathogenicity.