Clostridium difficile is a clinically important pathogen and the most common cause of hospital-acquired infectious diarrhea. Expression of the C. difficile gene csfV, which encodes σ(V), an extracytoplasmic function σ factor, is induced by lysozyme, which damages the peptidoglycan of bacteria. Here we show that σ(V) is required for lysozyme resistance in C. difficile. Using microarray analysis, we identified the C. difficile genes whose expression is dependent upon σ(V) and is induced by lysozyme. Although the peptidoglycan of wild-type C. difficile is intrinsically highly deacetylated, we have found that exposure to lysozyme leads to additional peptidoglycan deacetylation. This lysozyme-induced deacetylation is dependent upon σ(V). Expression of pdaV, which encodes a putative peptidoglycan deacetylase, was able to increase lysozyme resistance of a csfV mutant. The csfV mutant strain is severely attenuated compared to wild-type C. difficile in a hamster model of C. difficile-associated disease. We conclude that the σ(V) signal transduction system, which senses the host innate immune defense enzyme lysozyme, is required for lysozyme resistance and is necessary during C. difficile infection.