EbrAB in Bacillus subtilis belongs to a novel small multidrug resistance (SMR) family of multidrug efflux pumps. EmrE in Escherichia coli, a representative of SMR, functions as a homo-oligomer in the membrane. On the other hand, EbrAB requires a hetero-oligomeric configuration consisting of two polypeptides, EbrA and EbrB. Although both polypeptides have a high sequence similarity, expression of either single polypeptide does not confer the multidrug-resistance. We performed mutation studies on EbrA and B to determine why EbrAB requires the hetero-oligomerization. Mutants of EbrA and B lacking both the hydrophilic loops and the C-terminus regions conferred the multidrug-resistance solely by each protein. This suggests that the hydrophilic loops and the C-terminus regions constrain them to their respective conformations upon the formation of the functional hetero-oligomer.