Sertraline, an antidepressive drug, has been reported to inhibit general bacterial efflux pumps. In the present study, we report for the first time a synergistic effect of sertraline and tetracycline in a TetA-encoded tetracycline-resistant strain of Escherichia coli. Synergy between sertraline and tetracycline in an E. coli strain with TetA-mediated tetracycline resistance (E. coli APEC_O2) was assessed by the MIC and checkerboard assays. The global transcriptome of E. coli APEC_O2 exposed to ½ MIC concentrations of sertraline and/or tetracycline was analyzed to elucidate the interaction mechanism between sertraline and tetracycline. The fractional inhibitory concentration index for tetracycline and sertraline in E. coli APEC_O2 was 0.5. In addition, in the presence of ½ MIC of sertraline, the sensitivity of E. coli APEC_O2 to tetracycline could be restored according to clinical standards (from 64 to 4 mg l-1). RNA data suggest changes in respiration that is likely to decrease intracellular pH and thereby the proton-motive force, which provides the energy for the tetracycline efflux pump. Furthermore, sertraline and tetracycline may induce a change from oxidation to fermentation in the E.coli, which further decreases pH, resulting in cell death. This study shows that sertraline interacts with tetracycline in a synergistic and AcrAB-TolC pump-independent manner. The combinational treatment was further shown to induce many changes in the global transcriptome, including altered tetA and tetR expression. The results indicate that sertraline may be used as a helper compound with the aim to reverse tetracycline resistance encoded by tetA.