Clostridioides difficile infection (CDI) is a serious healthcare-associated disease, causing symptoms such as diarrhea. The main virulence factors responsible for the disease’s symptoms are two secreted cytotoxic proteins, toxin A (TcdA) and toxin B (TcdB). Treatment with antibiotics is difficult, as recurrent episodes of CDI usually occur in around 20-30% of patients after antibiotic therapy has ended. Several vaccines based on detoxified or recombinant forms of these toxins have been or are currently being tested in clinical trials. One of them is inactivated by formaldehyde, a method with obvious drawbacks. A new metal-catalyzed oxidation (MCO) method was used to detoxify the toxins while preserving the neutralizing epitopes. The aim of the project was therefore to combine these detoxified TxA and TxB toxins with a potent LiteVax adjuvant (LVA) to demonstrate vaccine safety and generate high levels of antibody responses in rabbits. For each study, 42 female rabbits, aged 8 weeks, were divided into 6 groups of 7 animals, each group vaccinated with different toxin concentrations (TxA between 5 and 20 µg and TxB between 20 and 80 µg) and different adjuvants (placebo, LVA adjuvant or alum). Each group was vaccinated three times at 14-day intervals by intramuscular route. Every day, the rabbits were observed for behavioral and feeding problems. Weekly kinetics were performed to determine serum and neutralizing antibody titers (against TcdA and TcdB), blood counts (cell populations) and inflammatory cytokines. White blood cell count and neutrophil percentage increased one day after immunization with LVA, but little or not with alum. Antibody titers were similar to those obtained with a conventional formaldehyde-inactivated CDI vaccine adjuvanted with alum. Alum enhanced antibody responses against TcdA but not against TcdB. LVA adjuvant generated significantly higher antibody responses against TcdB compared to alum. Histopathological analysis of the injection sites 42 days after the last injection showed no abnormal tissue reaction. In conclusion, MCO is an alternative method for the production of safe and immunogenic toxoid antigens, and TxA and TxB in combination with LVA adjuvant is a very interesting vaccine candidate against CDI.