Clostridium difficile is an important nosocomial pathogen that causes infectious diarrhea and pseudomembranous colitis among patients in hospitals worldwide and also poses a serious problem in farming animals. Recently, the incidence and severity of C. difficile infection has markedly increased due to resistance to many antibiotics. One of the mechanisms that have been associated with antibiotic resistance in C. difficile is through ATP binding-cassette (ABC) transporters which are well implicated in bacterial virulence and pathogenicity, making them potential targets for the development of new antimicrobial therapeutic strategies. The genome of C. difficile 630 was screened for sequences encoding putative ABC transporter proteins homologous to multidrug resistance ABC transporter in C. hathewayi. The putative ABC transporter gene was cloned into the pET28A expression vector and functionally expressed in Escherichia coli. The corresponding protein was detected and function of this protein was analyzed in antibiotic susceptibility assay. ABC transporter-expressing cells showed higher half minimal inhibitory concentration of antibiotics compared to the control cells. Therefore, ABC transporter might function as a multidrug transporter involved in multiple antibiotic resistance.