Multidrug efflux pumps belonging to the resistance-nodulation cell division (RND) family have major roles in the intrinsic and elevated resistance of Gram-negative bacteria to a wide range of compounds. RND efflux pumps require two other proteins to function: a membrane fusion protein (MFP) and an outer membrane protein. A recent study demonstrated that Salmonella enterica serovar Typhimurium has five RND efflux systems: AcrAB, AcrD, AcrEF, MdtABC and MdsABC. Most RND efflux system genes also code for an MFP in the same operon; however, an MFP gene is not located near acrD, and the MFP, with which AcrD functions, remains to be studied in detail. The aim of this study was to investigate the requirement of an MFP for the AcrD efflux system in this organism. When overproduced, AcrD significantly increased the resistance of the acrB mutant to oxacillin, cloxacillin, nafcillin, carbenicillin, sulbenicillin, aztreonam, sodium dodecyl sulfate and novobiocin. The increase in drug resistance caused by AcrD overproduction was completely suppressed by deleting the MFP gene, acrA, or the multifunctional outer membrane channel gene, tolC. Although the overexpression of acrD did not confer drug resistance to the ΔacrAB strain, co-overexpression of acrD with acrA increased the multidrug resistance of this mutant. Our results indicate that the AcrA MFP and TolC outer membrane protein, in addition to their roles in the AcrB efflux system, are required for the function of the AcrD efflux pump in S. enterica serovar Typhimurium.