Human respiratory mucus lining the airway epithelium forms a challenging barrier to inhalation therapeutics. Therefore, structural elucidation of hydrated mucus is essential for an efficient drug delivery development. The structure of mucus has been primarily investigated by conventional electron microscopy techniques, which operate under vacuum conditions and require sample preparation steps that might alter the structure of mucus. In this study we investigated the impact of dehydration on mucus and analyzed the structure of mucus in its hydrated state. Cryo-scanning electron microscopy (Cryo-SEM) analysis of mucus showed, that during the process of sublimation, non-porous structure of mucus is transformed into a porous network. Similarly, images acquired by environmental scanning electron microscopy (ESEM), revealed a non-porous structure of hydrated mucus, while further observation at decreasing pressure demonstrated the strong influence of dehydration on mucus structure. We could successfully visualize the structural organization of the major gel forming mucin MUC5B in its hydrated state by employing stimulated emission depletion (STED) microscopy, which allowed resolving the nano-scale patterns of mucin macromolecules within the essentially pore-free mucus structure. The general structural organization of mucus components was addressed by confocal laser scanning microscopy (CLSM), which revealed the heterogeneous and composite structure of mucus. These results provide a novel view on the native structure of mucus and will affect drug delivery development.