The rise of multidrug-resistant bacteria, such as Pseudomonas aeruginosa, jeopardizes public health, with increased fatalities among vulnerable individuals due to limited treatment options. P. aeruginosa's primary defense against antibiotics is the Resistance-Nodulation-Division (RND) membrane transport protein, specifically MexB in the MexAB-OprM complex. MexB's polyspecificity allows efflux of diverse molecules, including antibiotics. Through cryo-EM, we examined MexB's structure and ligand binding. We achieved a 2.16 Å resolution structure of MexB, revealing an asymmetric trimer where each unit has a distinct conformation: loose (L), tight (T), and open (O). Notably, the T-unit contains three DDM detergent molecules, with one in a previously unknown binding site. This challenges the belief that ligands shift binding sites during the L to T-state transition. 3D Variability Analysis (3DVA) suggests MexB is dynamic, undergoing significant conformational changes. We believe MexB possesses a resting (R) state related to the O-state and a new wide (W) state, with an expanded vestibule accommodating different substrate sizes. As the vestibule enlarges to the W-state, transmembrane helices seem to unfold, impacting MexB's bioenergetics due to the proton transport mechanism within these helices. In essence, our study provides a fresh structural insight into MexB's ligand binding and dynamism. Future research will compare this with clinical antibiotics to comprehend MexB efflux for potential inhibitor design.