Gram-negative bacteria export a large variety of antimicrobial compounds by forming two-membrane spanning tripartite multidrug efflux systems composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. Here we present the co-expression, purification and first electron microscopy insights of the Escherichia coli EmrAB–TolC tripartite Major Facilitator Superfamily (MSF) efflux system as a whole complex stabilized by Amphipol polymer. The structure reveals a 33 nm long complex delineated by the Amphipol belt at both extremities. Comparison of projection structures of EmrAB-TolC and AcrAB-TolC indicates that the outer membrane protein TolC linked to the periplasmic adaptor EmrA protein form an extended periplasmic canal. The overall length of EmrAB-TolC complex is similar to that of AcrAB-TolC with a probable tip-to-tip interaction between EmrA and TolC unveiling how the adaptor protein connects TolC and EmrB embedded in the inner membrane. Display omitted •Co-expression, mild extraction, and first EM insights of MFS-type EmrAB–TolC•EmrAB-TolC reveals a 33 nm long complex delineated by an Amphipol belt.•The overall length of EmrAB-TolC complex is similar to that of AcrAB-TolC.•There is a probable tip-to-tip interaction between EmrA and TolC.•TolC linked to the periplasmic adaptor EmrA forms an extended periplasmic canal.