ATP-binding cassette (ABC) transporters belong to one of the largest membrane protein superfamilies, which function in translocating substrates across biological membranes using energy from ATP hydrolysis. Currently, the classification of ABC transporters in Clostridioides difficile is not complete. Therefore, the sequence-function relationship of all ABC proteins encoded within the C. difficile genome was analyzed. Identification of protein domains associated with the ABC system in the C. difficile 630 reference genome revealed 226 domains: 97 nucleotide-binding domains (NBDs), 98 transmembrane domains (TMDs), 30 substrate-binding domains (SBDs), and one domain with features of an adaptor protein. Gene organization and transcriptional unit analyses indicated the presence of 78 ABC systems comprising 28 importers and 50 exporters. Based on NBD sequence similarity, ABC transporters were classified into 12 sub-families according to their substrates. Interestingly, all ABC exporters, accounting for 64% of the total ABC systems, are involved in antibiotic resistance. Based on analysis of ABC proteins from 49 C. difficile strains, the majority of core NBDs are predicted to be involved in multidrug resistance systems, consistent with the ability of this organism to survive exposure to an array of antibiotics. Our findings herein provide another step toward a better understanding of the function and evolutionary relationships of ABC proteins in this pathogen.