Hydroxyurea (HU), as a therapeutic medicine, has been extensively used clinically. To further survey molecular mechanisms of HU treatment, we analyzed global transcriptomic alteration of mouse ES cells in response to the treatment using high-throughput sequencing. We show that the global transcriptional activity is significantly suppressed as cells are exposed to HU treatment and alters multiple key cellular pathways, including cell cycle, apoptosis and DNAs. HU treatment also alters alternative splicing mechanisms and suppresses non-coding RNA expression. Our result provides novel clues for the understanding of how cells respond to HU and further suggests that high-throughput sequencing technology provides a powerful tool to study mechanisms of clinical drugs at the cellular level.