Numerous epigenetic alterations are observed in cancer cells, and dysregulation of mono-ubiquitination of histone H2B (H2Bub1) has often been linked to tumorigenesis. H2Bub1 is a dynamic post-translational histone modification associated with transcriptional elongation and DNA damage response. Histone H2B monoubiquitination occurs in the site of lysine 120, written predominantly by E3 ubiquitin ligases RNF20/RNF40 and deubiquitinated by ubiquitin specific peptidase 22 (USP22). RNF20/40 is often altered in the primary tumors including colorectal cancer, breast cancer, ovarian cancer, prostate cancer, and lung cancer, and the loss of H2Bub1 is usually associated with poor prognosis in tumor patients. The purpose of this review is to summarize the current knowledge of H2Bub1 in transcription, DNA damage response and primary tumors. This review also provides novel options for exploiting the potential therapeutic target H2Bub1 in personalized cancer therapy.