The global emergence of multidrug-resistant Gram-negative bacteria poses a significant threat to antibiotic therapy. Chromosomally encoded drug efflux mechanisms, particularly those represented by the AcrAB-TolC and Mex pumps of the resistance-nodulation-division (RND) superfamily, play a crucial role in antibiotic resistance. These pumps not only mediate intrinsic and acquired multidrug resistance (MDR) but also interact synergistically with other resistance mechanisms, such as the outer membrane permeability barrier, to enhance resistance levels. The discovery of RND pumps in the early 1990s has led to significant advancements in understanding their structural and biochemical basis, substrate profiles, molecular regulation, and inhibition. This review provides an up-to-date comprehensive description of efflux-mediated antibiotic resistance in Gram-negative bacteria, focusing on the biochemistry and genetics of multidrug efflux pumps, including the RND, MFS, MATE, SMR, and ABC superfamilies. The article also discusses the role of specific pumps, such as AcrB, in raising the minimum inhibitory concentrations (MICs) of antibiotics and the mechanisms of drug transport and efflux.The global emergence of multidrug-resistant Gram-negative bacteria poses a significant threat to antibiotic therapy. Chromosomally encoded drug efflux mechanisms, particularly those represented by the AcrAB-TolC and Mex pumps of the resistance-nodulation-division (RND) superfamily, play a crucial role in antibiotic resistance. These pumps not only mediate intrinsic and acquired multidrug resistance (MDR) but also interact synergistically with other resistance mechanisms, such as the outer membrane permeability barrier, to enhance resistance levels. The discovery of RND pumps in the early 1990s has led to significant advancements in understanding their structural and biochemical basis, substrate profiles, molecular regulation, and inhibition. This review provides an up-to-date comprehensive description of efflux-mediated antibiotic resistance in Gram-negative bacteria, focusing on the biochemistry and genetics of multidrug efflux pumps, including the RND, MFS, MATE, SMR, and ABC superfamilies. The article also discusses the role of specific pumps, such as AcrB, in raising the minimum inhibitory concentrations (MICs) of antibiotics and the mechanisms of drug transport and efflux.