Acylated amino acids function as important components of the cellular membrane in some bacteria. Biosynthesis is initiated by the -acylation of the amino acid, and this is followed by subsequent -acylation of the acylated molecule, resulting in the production of the mature diacylated amino acid lipid. In this study, we use both genetics and liquid chromatography-mass spectrometry (LC-MS) to characterize the biosynthesis and function of a diacylated glycine lipid (GL) species produced in We, and others, have previously reported the identification of a gene, named in this study, that encodes an -acyltransferase activity responsible for the production of a monoacylated glycine called -acyl-3-hydroxy-palmitoyl glycine (or commendamide). In all of the genomes sequenced so far, the gene is located immediately downstream from a gene, named , that is also predicted to encode a protein with acyltransferase activity. We use LC-MS to show that the coexpression of and results in the production of GL in We constructed a deletion mutant of the gene in , and we confirm that is required for the production of GL in Moreover, we show that is important for the ability of to adapt to stress and colonize the mammalian gut. Therefore, this report describes the genetic requirements for the biosynthesis of GL, a diacylated amino acid species that contributes to fitness in the human gut bacterium The gut microbiome has an important role in both health and disease of the host. The mammalian gut microbiome is often dominated by bacteria from the , an order that includes and In this study, we have identified an acylated amino acid, called glycine lipid, produced by , a beneficial bacterium originally isolated from the human gut. In addition to identifying the genes required for the production of glycine lipids, we show that glycine lipids have an important role during the adaptation of to a number of environmental stresses, including exposure to either bile or air. We also show that glycine lipids are important for the normal colonization of the murine gut by This work identifies glycine lipids as an important fitness determinant in and therefore increases our understanding of the molecular mechanisms underpinning colonization of the mammalian gut by beneficial bacteria.