Clostridioes difficile is a Gram-positive, spore-forming, obligate anaerobic bacterium, and the main cause of hospital-associated diarrhea. In recent years, with the presence of virulent strains (i.e., ribosome type 027), the prevalence and mortality events have increased. Thus, studies on physiological and biochemical characteristics, and pathogenic mechanisms of C. difficile have been performed. The development of efficient and stable genome-editing methods for C. difficile is urgent for the dissection of its physiological and pathogenic mechanism. For example, ClosTron technology plays a key role in study of the relationship between C. difficile toxins (Toxin A and Toxin B) and its pathogenicity. This article reviews the history, recent progress and future prospects of C. difficile genome-editing technologies.