Clostridium difficile infection is the major cause of nosocomial diarrhea and colitis in developed countries. The broad use of antibiotics has contributed to the occurrence and spread of antibiotic-resistant C. difficile strains in recent years. The main virulent factors of its pathogenicity are two secreted cytotoxins, Toxin A and Toxin B, which attack the intestinal epithelium cell barriers and cause follow-up inflammation and bowel damage. Here, truncated fragments of the receptor binding domain of C. difficile Toxin A, which are responsible for the binding to receptors on colonic epithelial cells and facilitating internalization, endocytosis, and entry into the cell cytoplasm, were used for the design of mRNA vaccines against C. difficile. The mRNA-encoded bacterial antigens were found to be able to successfully express in vitro in mammalian cells. Mice were injected with 3 doses of the mRNA vaccine and the immunogenicity was evaluated by ELISA for the detection of high-titer toxin-specific antibodies. The induced immune serum was able to neutralize native whole C. difficile Toxin A and protect Vero cells from death in vitro. This study can provide a framework for other antibacterial mRNA vaccines in the future.