Molecular dynamics simulations provide essential atomistic insights into the organization and dynamics of complex biological membranes. However, the equilibration of large-scale, curved lipid assemblies at the all-atom level remains a significant challenge. Standard construction approaches, such as wrapping planar bilayers onto spherical meshes, frequently reverberate into structural instabilities, including membrane holes, infiltrated water, lipid flipping, and nonequilibrated densities, which hinder stable production simulations. Here, we present ReVesicle, an iterative equilibration protocol designed to restore and stabilize quasi-spherical lipid vesicles with complex compositions and large dimensions. The proposed protocol combines selective identification and removal of infiltrated water molecules and flipped lipids with short nonequilibrium MD cycles and anisotropic pressure equilibration. These steps are organized into a modular, iterative sequence that progressively recovers bilayer continuity while preserving the vesicle geometry and enabling global density relaxation. Local vacuum-induced stress generated during nonequilibrium phases promotes lipid tail melting and hole curation, while anisotropic equilibration allows relaxation of box dimensions and system density. To demonstrate the robustness of ReVesicle, we applied the protocol to six biologically realistic vesicle systems: synaptic vesicles, plasma membranes, late endosomes, exosomes, mitochondria-derived vesicles, and the HIV-1 lipid envelope. These systems span diameters from 40 to 105 nm and reach total sizes of up to ∼150 million atoms with heterogeneous and asymmetric lipid compositions. Across all cases, ReVesicle consistently converges to continuous, tightly packed bilayers. Structural and biophysical analyses, including vesicle diameter, sphericity, area per lipid, and lipid acyl-chain order parameters, indicate preservation of quasi-spherical geometry and structural integrity. Overall, ReVesicle provides a reproducible framework for equilibrating large, heterogeneous lipid vesicles suitable for downstream all-atom simulations of complex biological environments.

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