TetA(B), a tetracycline efflux protein from Tn10 with 12 predicted transmembrane α-helices and belonging to the major facilitator superfamily, uses the bacterial transmembrane proton motive force to export metal-tetracycline complexes in exchange for H⁺. Previous studies showed that the cytoplasmic interdomain loop connecting transmembrane helices 6 and 7 of TetA(B) contains residues involved in tetracycline efflux activity, and Asp190, Glu192, and Ser201 are implicated in substrate specificity. In this study, we investigated the effect of the Asp190Cys mutation on TetA(B)-mediated tetracycline efflux by transforming Escherichia coli DH5α with low-copy plasmids (pSC101 origin) carrying wild-type or mutant TetA(B), preparing everted membrane vesicles, and using [³H]tetracycline uptake assays, along with MIC determination for intact cells, Western immunoblotting to verify consistent TetA(B) protein levels in membranes, and Graphpad Prism 4 software to fit Km and Vmax values via the Michaelis-Menten equation. Results showed that the Asp190Cys mutant had an average Km value 3.8 times that of the wild type (83 ± 24 μM vs. 22 ± 6 μM) without modifying Vmax (1327 ± 187 pmol/mg protein/min vs. 1274 ± 106 pmol/mg protein/min for wild type). MIC assays revealed reduced tetracycline resistance in intact DH5 cells expressing the mutant (MIC = 11 μg/ml) compared to the wild type (MIC = 128 μg/ml). These findings indicate that the Asp190Cys mutation decreases TetA(B)'s affinity for tetracycline, likely due to the loss of the negatively charged aspartate that interacts with the positively charged metal-tetracycline complex. Furthermore, our biochemical results support that the cytoplasmic interdomain loop, previously thought to merely tether the two halves of the protein, plays an unexpected role in tetracycline transport.