Ubiquitin is best known for targeting protein degradation by the 26 S proteasome. In recent years, however, roles of ubiquitin are found far more than these. Ubiquitin not only has "traditional function" participating proteins degradation, but also plays a more varied and decisive role in cellular regulation than previously imagined. It is a multilayer regulator of important cellular processes and has a great many nonproteolytic functions including DNA damage repair, DNA replication, signal transduction, transcriptional regulation, membrane trafficking, endocytosis, protein kinase activation, chromatin remodeling and virus budding. These functions involve polyubiquitylation, monoubiquitylation and multiubiquitylation. Therefore, abnormities in ubiquitylation involves occurrence and development of diseases. Understanding these functions will provide further insights into the repertoire of ubiquitin, help to understand diverse cellular processes and facilitate our development of related new drugs.