Biological membranes form the functional, dynamic interface that hosts a major fraction of all cellular bioactivity. Proper membrane physiology requires maintenance of a narrow range of physicochemical properties, which must be buffered from external perturbations. While homeostatic adaptation of membrane fluidity to temperature variation is a ubiquitous design feature of ectothermic organisms, such responsive membrane adaptation to external inputs has not been directly observed in mammals. Here, we report that challenging mammalian membrane homeostasis by dietary lipids leads to robust lipidomic remodeling to preserve membrane physical properties. Specifically, exogenous polyunsaturated fatty acids (PUFAs) are rapidly and extensively incorporated into membrane lipids, inducing a reduction in membrane packing. These effects are rapidly compensated both in culture and
in vivoby lipidome-wide remodeling, most notably upregulation of saturated lipids and cholesterol. These lipidomic changes result in recovery of membrane packing and permeability. This lipidomic and biophysical compensation is mediated in part by lipid regulatory machinery, whose pharmacological or genetic abrogation results in cytotoxicity when membrane homeostasis is challenged by dietary lipids. These results reveal an essential mammalian mechanism for membrane homeostasis wherein lipidome remodeling in response to dietary lipid inputs preserves functional membrane phenotypes.