Updated project metadata. Osteogenesis is the process of bone formation and is modulated by multiple regulatory networks. With the rapid development of the epitranscriptomics field, RNA modifications and their reader, writer, and eraser (RWE) proteins are shown to be involved in the regulation of various biological processes. Few studies, however, were conducted to investigate the functions of RNA modifications and their RWE proteins in osteogenesis. By using a parallel-reaction monitoring (PRM)-based targeted proteomics method, we performed a comprehensive quantitative assessment of 154 epitranscriptomic RWE proteins during the time course of osteogenic differentiation of H9 human embryonic stem cells (ESCs). We found that approximately half of the 126 detected RWE proteins were downregulated during osteogenic differentiation, and they included mainly those proteins involved in RNA methylation and pseudouridine synthesis. Protein-protein interaction (PPI) network analysis revealed a high connectivity between the downregulated epitranscriptomic RWE proteins and osteogenesis-related proteins. Gene set enrichment analysis of previously published RNA-seq data from osteogenesis imperfecta patients suggested a potential role of METTL1, the top-ranked hub protein of downregulated RWE proteins, in osteogenesis through the cytokine network. Together, this is the first targeted profiling of epitranscriptomic RWE proteins during osteogenic differentiation of human ESCs and our work unveiled potential regulatory roles of these proteins in osteogenesis.