Multidrug efflux pumps contribute to the resistance of Escherichia coli against many antibiotics and biocides. Here, we report that the CRP regulator modulates multidrug resistance in E. coli through repression of the genes encoding the MdtEF multidrug efflux pump. Screening of mutants for ability to increase beta-lactam resistance in E. coli led to the identification of a mutation in crp, which codes for the major global regulator of catabolite-sensitive operons. Deletion of crp significantly increased the resistance of the E. coli strain to oxacillin, azithromycin, erythromycin and crystal violet. The increase in drug resistance caused by crp deletion was completely suppressed by deletion of the multifunctional outer membrane channel gene tolC. TolC interacts with different drug efflux pumps. Among the twenty drug efflux pumps in E. coli, quantitative real-time PCR analysis showed that CRP repressed the expression of mdtEF. Deletion of mdtEF completely suppressed CRP-modulated multidrug resistance. Therefore, in addition to its role in catabolite control, CRP contributes to multidrug resistance in E. coli. Our results indicate that the CRP regulator modulates multidrug resistance in E. coli by repressing expression of the MdtEF multidrug efflux pump.