Protein modification by ubiquitin is one of the most versatile posttranslational regulations and counteracted by almost 100 deubiquitinating enzymes (DUBs). USP8 was originally identified as a growth regulated ubiquitin-specific protease and is like many other DUBs characterized by its multidomain architecture. Besides the catalytic domain, specific protein–protein interaction modules were characterized which contribute to USP8 substrate recruitment, regulation and targeting to distinct protein complexes. Studies in mice and humans impressively showed the physiological relevance and non-redundant function of USP8 within the context of the whole organism. USP8 knockout (KO) mice exhibit early embryonic lethality while induced deletion in adult animals rapidly causes lethal liver failure. Furthermore, T-cell specific ablation disturbs T-cell development and function resulting in fatal autoimmune inflammatory bowel disease. In human patients, somatic mutations in USP8 were identified as the underlying cause of adrenocorticotropic hormone (ACTH) releasing pituitary adenomas causing Cushing's disease (CD). Here we provide an overview of the versatile molecular, cellular and pathology associated function and regulation of USP8 which appears to depend on specific protein binding partners, substrates and the cellular context.