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The insertional inactivation of<jats:italic>dltD<\/jats:italic>of the probiotic strain<jats:italic>Lactobacillus rhamnosus<\/jats:italic>GG (ATCC 53103) resulted in the complete absence of<jats:sc>d<\/jats:sc>-alanyl esters in the LTA as confirmed by nuclear magnetic resonance analysis. This was reflected in modifications of the bacterial cell surface properties. The<jats:italic>dltD<\/jats:italic>strain showed 2.4-fold-increased cell length, a low survival capacity in response to gastric juice challenge, an increased sensitivity to human beta-defensin-2, an increased rate of autolysis, an increased capacity to initiate growth in the presence of an anionic detergent, and a decreased capacity to initiate growth in the presence of cationic peptides compared to wild-type results. However, in vitro experiments revealed no major differences for adhesion to human intestinal epithelial cells, biofilm formation, and immunomodulation. 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