Adenoviruses enter host cells through receptor-mediated endocytosis and acid-activated penetration from endosomes into the cytosol, where they deliver their DNA genome into the nucleus. This study demonstrates that incoming adenovirus type 2 particles undergo a stepwise disassembly process necessary for the progression of the viral entry pathway and the release of the genome into the nucleus. The disassembly begins immediately upon endocytic uptake, involving the release of fibers, dissociation of penton base structures, degradation or shedding of proteins connecting the DNA to the inside surface of the capsid, and elimination of minor capsid-stabilizing proteins. The uncoating process starts with the loss of fibers and ends with the uptake of dissociated hexon proteins and DNA into the nucleus. Key events include the proteolysis of protein VI and the dissociation of the penton base complex, which occur within 20 minutes of endocytosis. The study also reveals that the fiber protein is essential for virus endocytosis and that the disassembly process is highly efficient, with about 40% of initially bound viruses releasing their DNA inside the cell.Adenoviruses enter host cells through receptor-mediated endocytosis and acid-activated penetration from endosomes into the cytosol, where they deliver their DNA genome into the nucleus. This study demonstrates that incoming adenovirus type 2 particles undergo a stepwise disassembly process necessary for the progression of the viral entry pathway and the release of the genome into the nucleus. The disassembly begins immediately upon endocytic uptake, involving the release of fibers, dissociation of penton base structures, degradation or shedding of proteins connecting the DNA to the inside surface of the capsid, and elimination of minor capsid-stabilizing proteins. The uncoating process starts with the loss of fibers and ends with the uptake of dissociated hexon proteins and DNA into the nucleus. Key events include the proteolysis of protein VI and the dissociation of the penton base complex, which occur within 20 minutes of endocytosis. The study also reveals that the fiber protein is essential for virus endocytosis and that the disassembly process is highly efficient, with about 40% of initially bound viruses releasing their DNA inside the cell.