Abstract: Eukaryotic gene regulation is closely correlated with histone covalent modifications. Recently, histone H2B lysine-123 (H2B-K123) ubiquitination has been implicated in regulation of transcription as well as histone H3 lysine-4 (H3-K4) methylation which is further associated with active transcription. However, whether H2B-K123 ubiquitination controls transcription through regulation of H3-K4 methylation remains unknown under physiological conditions. Here, we show that H2B-K123 ubiquitination enhances the rate of elongating RNA polymerase II (RNAPII) recruitment to the coding sequence of an inducible yeast gene, GAL1. Consistently, GAL1 transcription is significantly impaired in absence of H2B-K123 ubiquitination. On the other hand, H3-K4 methylation does not alter the rate of elongating RNAPII recruitment at GAL1. Further, these covalent modifications do not regulate pre-initiation complex formation at GAL1. Collectively, our data demonstrate the function of H2B-K123 ubiquitination in regulation of transcriptional elongation independently of H3-K4 methylation in vivo, providing a new insight on epigenetic regulation of gene expression.