The Mediator complex orchestrates multiple transcription factors with the Pol Ⅱ apparatus for precise transcriptional control.However, its interplay with the surrounding chromatin remains poorly understood.Here, we analyze differential histone modifications between WT and MED23-/-(KO) cells and identify H2B mono-ubiquitination at lysine 120 (H2Bub) as a MED23-dependent histone modification.Using tandem affinity purification and mass spectrometry, we find that MED23 associates with the RNF20/40 complex, the enzyme for H2Bub, and show that this association is critical for the recruitment of RNF20/40 to chromatin.In a cell-free system, Mediator directly and substantially increases H2Bub on recombinant chromatin through its cooperation with RNF20/40 and the PAF complex.Integrative genome-wide analyses show that MED23 depletion specifically reduces H2Bub on a subset of MED23-contolled genes.Importantly, MED23-coupled H2Bub levels are oppositely regulated during myogenesis and lung carcinogenesis.In sum, these results establish a mechanistic link between the Mediator complex and a critical chromatin modification in coordinating transcription with cell growth and differentiation.