Drug-resistance dissemination by horizontal gene transfer remains poorly understood at the cellular scale. Using live-cell microscopy, we reveal the dynamics of resistance acquisition by transfer of the Escherichia coli fertility factor–conjugation plasmid encoding the tetracycline-efflux pump TetA. The entry of the single-stranded DNA plasmid into the recipient cell is rapidly followed by complementary-strand synthesis, plasmid-gene expression, and production of TetA. In the presence of translation-inhibiting antibiotics, resistance acquisition depends on the AcrAB-TolC multidrug efflux pump, because it reduces tetracycline concentrations in the cell. Protein synthesis can thus persist and TetA expression can be initiated immediately after plasmid acquisition. AcrAB-TolC efflux activity can also preserve resistance acquisition by plasmid transfer in the presence of antibiotics with other modes of action