Multidrug resistance (MDR) efflux pumps are increasingly recognized as major contributors to resistance in clinically resistant isolates. MDR is associated with increased morbidity and mortality in hospitals and is a serious public health concern. The major MDR pumps in Gram-negative bacteria belong to members of the resistance-nodulation-cell division (RND) family. RND pumps function as tripartite complexes, together with an outer membrane (OM) channel and a periplasmic membrane fusion protein (MFP). The MFP is proposed to act as a bridge between the inner membrane (IM) transporter and the OM channel thus allowing the direct efflux of drugs across the two-membrane cell envelope of Gram-negative bacteria. MFPs also function with transporters belonging to ATP binding Cassette (ABC) family or major facilitator superfamily (MFS) in the efflux of diverse substrates from small molecules to peptide toxin. The precise role of MFPs in the transport process remains elusive. Structural homologues of MFPs are also identified in Gram-positive bacteria that do not have a two-membrane cell envelope. These findings suggest that MFPs may play additional role other than that of a structural linker. Our studies with reconstituted systems of MFP-dependent ABC transporters, MacAB-TolC and YknXYZ from Escherichia coli and Bacillus subtilis respectively, revealed that MFPs play an active functional role by stimulating the ATPase activity of its cognate transporter. Consistent with previous studies, we found that the C-terminal domain of MFPs to be important for forming a functional interaction with the transporter. Our results also suggest that MFPs function through similar mechanism in both Gram-negative and Gram-positive bacteria.