Protein phosphorylation is central to the understanding of multiple cellular signaling pathways responsible for regulating the self-renewal and differentiation of neural stem cells (NSCs). Characterization of phosphoproteome is the key to unraveling the underlying mechanism. Here we performed a large-scale phosphoproteomic analysis of rat fetal NSCs (rfNSCs) utilizing strong cation exchange chromatography (SCX) prefractionation and citric acid-assisted two-step enrichment with TiO2 (CATSET) strategy followed by nanoLC-MS/MS analysis. Totally we identified 11,629 phosphosites on 3,046 phosphoproteins, among which 8,146 were class I phosphosites. More than 40% of class I phosphosites were novel when compared with PhosphoSitePlus database. Those phosphorylated proteins were implicated in modulation of diverse nuclear processes, such as transcription regulation, chromatin organization and chromatin modifications. Remarkably, 86 novel phosphosites on 43 transcriptional regulators were identified for the first time, including novel phosphorylation sites on Sox2 (S39) and Sox6 (S439, S442). Furthermore, bioinformatics analysis suggested that a large set of phosphosites were present on proteins involved in diverse pivotal signaling pathways associated with NSCs maintenance, and high degree of phosphorylation in insulin and Wnt signaling pathway was revealed. To our knowledge, this study presents the largest global survey of phosphorylation modification which revealed new regulation network in rfNSCs for the first time and may provide a valuable resource for studying the self-renewal and differentiation of NSCs.