Membrane proteins (MPs), encoded by \~30% of the coding genes, play vital roles in numerous physiological processes. MPs are targets of more than half of the FDA-approved drugs. High-resolution structural studies of functional membrane proteins under near-physiological conditions are required to provide an in-depth mechanistic understanding and to facilitate drug discovery. To embed the proteins into liposomes represents a strategy to mimic native membrane conditions. Here we present a convenient workflow for cryo-EM analysis of liposome-embedded MPs using the prototypal protein AcrB. Our method sets the foundation for future investigation of MPs in the presence of electrochemical gradients and for the understanding of the interdependence of integral or peripheral MPs and various membrane properties.Membrane proteins (MPs) used to be the most difficult targets for structural biology when X-ray crystallography was the mainstream approach. With the resolution revolution of single-particle electron cryo-microscopy (cryo-EM), rapid progress has been made for structural elucidation of isolated MPs. The next challenge is to preserve the electrochemical gradients and membrane curvature for a comprehensive structural elucidation of MPs that rely on these chemical and physical properties for their biological functions. Toward this goal, here we present a convenient workflow for cryo-EM structural analysis of MPs embedded in liposomes, using the well-characterized AcrB as a prototype. Combining optimized proteoliposome isolation, cryo-sample preparation on graphene grids, and an efficient particle selection strategy, the three-dimensional (3D) reconstruction of AcrB embedded in liposomes was obtained at 3.9 Å resolution. The conformation of the homotrimeric AcrB remains the same when the surrounding membranes display different curvatures. Our approach, which can be widely applied to cryo-EM analysis of MPs with distinctive soluble domains, lays out the foundation for cryo-EM analysis of integral or peripheral MPs whose functions are affected by transmembrane electrochemical gradients or/and membrane curvatures.