Ubiquitination is an important post-translational modification that has a pivotal role in numerous biological functions, such as cell growth, proliferation, apoptosis, DNA damage response, innate immune response and neuron degeneration. Although ubiquitination is thought to achieve these functions by targeting proteins for proteasome-dependent degradation, recent studies suggest that ubiquitination also has nonproteolytic functions, such as protein trafficking, kinase and phosphatase activation, which are involved in cell survival and cancer development. These progresses have advanced our current understanding of the novel functions of ubiquitination in signal transduction pathways and may provide novel paradigms for the treatment of human cancers.