Biofilm production is thought to be an important step in many enterococcal infections. In several Gram-positive bacteria, membrane glycolipids have been implicated in biofilm formation. We constructed a non-polar deletion mutant of a putative glucosyltransferase designated biofilm-associated glycolipid synthesis A (bgsA) in Enterococcus faecalis 12030. Analysis of major extracted glycolipids by nuclear magnetic resonance spectroscopy revealed that the cell membrane of 12030 Delta bgsA was devoid of diglucosyl-diacylglycerol (DGlcDAG), while monoglucosyl-diacylglycerol was overrepresented. The cell walls of 12030 Delta bgsA contained longer lipoteichoic acid molecules and were less hydrophobic than wild-type bacteria. Inactivation of bgsA in E. faecalis 12030 and E. faecalis V583 led to an almost complete arrest of biofilm formation on plastic surfaces. Overexpression of bgsA, on the other hand, resulted in increased biofilm production. While initial adherence was not affected, bgsA-deficient bacteria did not accumulate in the growing biofilm. Also, adherence of E. faecalis Delta bgsA to Caco-2 cells was impaired. In a mouse bacteraemia model, E. faecalis 12030 Delta bgsA was cleared more rapidly from the bloodstream than the wild-type strain. In summary, BgsA is a glycosyltransferase synthetizing DGlcDAG, a glycolipid and lipoteichoic acid precursor involved in biofilm accumulation, adherence to host cells, and virulence in vivo.