Histone ubiquitination plays a critical role in the regulation of transcription, and histone H2B monoubiquitination (H2Bub1) is mainly associated with transcriptional activation. Recent studies in yeast, humans, and Arabidopsis have revealed the conservation of chromatin modification via H2Bub1 during evolution. Rad6-Bre1 and their homologs are responsible for H2B monoubiquitination in diverse eukaryotic organisms, and the PAF complex is required for H2Bub1 to proceed. H2Bub1 is involved in many developmental processes in yeast, humans, and Arabidopsis, and it activates gene transcription by regulating the H3K4 methylation state. Notably, the level of H3K4 methylation is entirely dependent on H2Bub1 in yeast and humans, whereas the H3K4 methylation level of only a small number of genes in Arabidopsis is dependent on H2Bub1. In this review, we summarize the enzymes involved in H2B monoubiquitination and deubiquitination, and discuss the biologic functions of H2Bub1 in different organisms. In addition, we focus on recent advances in our understanding of the molecular mechanisms that enable H2Bub1 to perform its function.