Oxygen availability governs the energy homeostasis and development of living organisms. Cellular adaptation to the changes of the oxygen concentration are regulated by diverse signaling and transcriptional mechanisms. Proline trans-4-hydroxylation (Hyp) is an evolutionarily conserved posttranslational modification (PTM) directly involved in oxygen sensing. Using affinity-based proteomics analysis, we performed the first systematic profiling of proline hydroxylation targets. Our bioinformatic analysis revealed the functional importance of the modification in protein secondary structure and protein-protein interaction. Our study identified BRD4, a key transcription factor, as a novel proline hydroxylation substrate. Application of targeted mass spectrometry and quantitative interactome proteomic analysis determined the regulatory pathway and functional significance of BRD4 proline hydroxylation. Phenotypic analysis further revealed the translational potential of targeting the oncogenic BRD4 proline hydroxylation in leukemia.