LpxC, the deacetylase that catalyzes the second and committed step of lipid A biosynthesis in Escherichia coli, is an essential enzyme in virtually all gram-negative bacteria and is one of the most promising antibiotic targets for treatment of multidrug-resistant gram-negative infections. Despite the rapid development of LpxC-targeting antibiotics, the potential mechanisms of bacterial resistance to LpxC inhibitors remain poorly understood. Here, we report the isolation and biochemical characterization of spontaneously arising E. coli mutants that are over 200-fold more resistant to LpxC inhibitors than the wild-type strain. These mutants have two chromosomal point mutations that account for resistance additively and independently; one is in fabZ, a dehydratase in fatty acid biosynthesis; the other is in thrS, the Thr-tRNA ligase. For both enzymes, the isolated mutations result in reduced enzymatic activities in vitro. Unexpectedly, we observed a decreased level of LpxC in bacterial cells harboring fabZ mutations in the absence of LpxC inhibitors, suggesting that the biosyntheses of fatty acids and lipid A are tightly regulated to maintain a balance between phospholipids and lipid A. Additionally, we show that the mutation in thrS slows protein production and cellular growth, indicating that reduced protein biosynthesis can confer a suppressive effect on inhibition of membrane biosynthesis. Altogether, our studies reveal a previously unrecognized mechanism of antibiotic resistance by rebalancing cellular homeostasis.