The dlt operon encodes proteins responsible for the esterification of positively charged D-alanine on the wall teichoic acids and lipoteichoic acids of Gram-positive bacteria. This structural modification of the bacterial anionic surface in several species has been described to alter the physicochemical properties of the cell-wall. In addition, it has been linked to reduced sensibilities to cationic antimicrobial peptides and antibiotics. We studied the D-alanylation of Clostridioides difficile polysaccharides with a complete deletion of the dltDABC operon in the 630 strain. To look for D-alanylation location, surface polysaccharides were purified and analyzed by NMR. Properties of the dltDABC mutant and the parental strains, were determined for bacterial surface’s hydrophobicity, motility, adhesion, antibiotic resistance. We confirmed the role of the dltDABC operon in D-alanylation. Then, we established the exclusive esterification of D-alanine on C. difficile lipoteichoic acid. Our data also suggest that D-alanylation modifies the cell-wall’s properties, affecting the bacterial surface’s hydrophobicity, motility, adhesion to biotic and abiotic surfaces,and biofilm formation. In addition, our mutant exhibitedincreased sensibilities to antibiotics linked to the membrane, especially bacitracin. A specific inhibitor DLT-1 of DltA reduces the D-alanylation rate in C. difficile but the inhibition was not sufficient to decrease the antibiotic resistance against bacitracin and vancomycin. Our results suggest the D-alanylation of C. difficile as an interesting target to tackle C. difficile infections.