The Gram-positive lactic acid bacterium Lactobacillus buchneri CD034 is covered by a two-dimensional crystalline, glycoproteinaceous cell surface (S-) layer lattice. While lactobacilli are extensively exploited as cell surface display systems for applied purposes, questions about how they stick their cell wall together are remaining open. This also includes the identification of the S-layer cell wall ligand. In this study, lipoteichoic acid was isolated from the L. buchneri CD034 cell wall as a significant fraction of the bacterium's cell wall glycopolymers, structurally characterized and analyzed for its potential to mediate binding of the S-layer to the cell wall. Combined component analyses and 1D- and 2D-nuclear magnetic resonance spectroscopy (NMR) revealed the lipoteichoic acid to be composed of on average 31 glycerol-phosphate repeating units partially substituted with α-d-glucose, and with an α-d-Galp(1→2)-α-d-Glcp(1→3)-1,2-diacyl-sn-Gro glycolipid anchor. The specificity of binding between the L. buchneri CD034 S-layer protein and purified lipoteichoic acid as well as their interaction force of about 45 pN were obtained by single-molecule force spectroscopy; this value is in the range of typical ligand-receptor interactions. This study sheds light on a functional implication of Lactobacillus cell wall architecture by showing direct binding between lipoteichoic acid and the S-layer of L. buchneri CD034.