Significance The 26S proteasome is a giant protease assembled from at least 32 different canonical subunits. In eukaryotic cells it is responsible for the regulated degradation of proteins marked for destruction by polyubiquitin tags. Mainly because of the conformational heterogeneity of the 26S holocomplex, its structure determination has been challenging. Using cryo-electron microscopy single-particle analysis we were able to obtain a high-resolution structure of the human 26S proteasome allowing us to put forward an essentially complete atomic model. This model provides insights into the proteasome’s mechanism of operation and could serve as a basis for structure-based drug discovery.