D-alanylation of surface polysaccharides reduces the affinity and efficacy of cationic antimicrobial compounds (CAMPs) on the bacterial surface. In C. difficile (CD), the localization of D-alanylation is unknown and its implication in antibiotic resistance is not elucidated. The aim of our study is to determine the site of D-alanylation in CD and investigate its role in antibiotic susceptibility. A Δdlt mutant was first constructed. The two major CD polysaccharides, type II polysaccharide (PSII) and lipoteichoic acid (LTA) from the Δdlt mutant and its parental strain were purified. NMR analysis of the polysaccharides revealed the presence of D-alanine on the LTA of the parental strain, but not on the LTA of the Δdlt mutant. In addition, no D-alanine function was identified on purified PSII. Our results therefore highlight the exclusive D-alanylation of LTA in CD. The Δdlt mutant has multiple phenotypes, including greater surface hydrophobicity, increased motility, decreased adherence and increased biofilm formation than the parental strain. In addition, antibiotic susceptibility tests have noteworthy shown increased sensitivity of the Δdlt mutant to bacitracin, teicoplanin and daptomycin. The Dlt1 inhibitor, targeting DltA and already described in Staphylococcus aureus and Enterrococci, was also tested in CD. Our results support the interest in D-alanylation as a potential therapeutic target in the treatment of CD infections.