Ubiquitin, a highly conserved 76-amino-acid polypeptide widely present in eukaryotic cells, is covalently attached to substrate proteins through the E1-E2-E3 cascade. Ubiquitination is involved in a myriad of cellular functions, including protein degradation, signal transduction, DNA damage and repair, transcription regulation, cell cycle progression, and tumorigenesis. In this review, we discuss the basic concept of protein ubiquitination, and focus on its roles in the initiation and progression of cancers, DNA damage response, and neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease. These knowledge is staring to be exploited to develop both drugs and therapy strategies against these diseases.