Expelling heterologous compounds out of hosts by transporters is a potential strategy to enhance product titers in microbial cell factories. In this work, to increase heterologous polyketide 6-deoxyerythronolide B (6dEB, erythromycin precursor) production, tripartite multidrug efflux pumps MacAB-TolC, AcrAB-TolC, MdtEF-TolC, and MexAB-OprM were modulated in a 6dEB production strain. Compared with the control, overexpression of a single component of efflux pumps (except oprM) repressed 6dEB production, but modulation of two components MacA and MacB or the complete pumps MacAB-TolC and MdtEF-TolC significantly improved 6dEB titer by 100 ± 11, 118 ± 54, and 98 ± 12 %, respectively. In addition, to avoid the challenging fine-tuning components of pumps, the transcriptional regulators of efflux pumps were modulated to improve the 6dEB production. Overexpression of RpoH (activator of MdtEF-TolC) and EvgA (activator of EmrKY-TolC and AcrAD-TolC) strongly increased 6dEB titer by 152 ± 54 and 142 ± 85 %, respectively. This is the first report of transporter engineering for improving heterologous polyketide production in Escherichia coli. Our results provide an effective strategy for improving the yield of the heterologous products in chassis cell.