Clostridioides difficile is the leading cause of antibiotics-associated diarrhea but can also result in more serious, life-threatening conditions. As incidence of C. difficile infections in hospitals is increasing, both in frequency and severity, and antibiotic-resistant C. difficile strains are advancing, antimicrobial peptides (AMPs) are an interesting alternative to classic antibiotics. Knowledge on the effects of AMPs on C. difficile will therefore not only enhance the knowledge for possible biomedical application but may also provide insights in mechanisms of C. difficile to adapt or counteract AMPs. This study applies state-of-the-art mass spectrometry methods to quantitatively investigate the proteomic response of C. difficile 630∆erm to sublethal concentrations of the AMP nisin allowing to follow the cellular stress adaptation in a time-resolved manner. The results do not only point at a heavy reorganization of the cellular envelop but also determined pronounced changes in central cellular processes such as carbohydrate metabolism. Moreover, the number of flagella per cell was changed during the process of adaptation to nisin suggesting a role of these cellular structures in combating this particular AMP, which is independent from pure cell motility