17β-Estradiol (E2) regulates transcriptional activity partly by inducing protein-kinase cascades, leading to the phosphorylation of estrogen receptors (ERs) and other functional proteins. Many of these phosphorylation events are also modulated by growth factors. To gain an insight into E2-modulated protein phosphorylation, we applied quantitative phosphoproteomics to investigate global changes in protein phosphorylation induced by E2 in MCF-7 cells. Proteomic analyses using stable isotope dimethyl labeling coupled with immobilized metal affinity chromatography-hydrophilic interaction liquid chromatography (IMAC-HILIC) fractionation and nanoLC-MS/MS identified and quantified 2857 unique phosphorylation sites in 1338 phosphoproteins from 1 mg of total cellular protein. In addition to S118 of ERα, a 30-min E2 treatment significantly altered the status of 403 phosphorylation sites, including 112 novel sites. Interestingly, the substrate motifs for ERK1/2 were largely enriched in both the up-regulated and down-regulated phosphorylation sites. An increase in the phosphorylation on either the T202 or Y204 sites of ERK1 was observed after E2 treatment, while dual phosphorylation on both sites were not detected, implying that a feedback loop to deactivate MAPK signaling was achieved during a 30-min E2 treatment. In contrast, the PKA and CKII substrate motifs were majorly enriched among the up-regulated phosphorylation sites. Western blot analysis confirmed that E2 increased the phosphorylation level of S226 within a CKII motif of HSP90β by a factor of 2- to 3-fold without changing the total protein expression level. E2 also up-regulated phosphorylations of S255 in HSP90β and S353 within a CKII motif of HSP90α. These results indicated that E2 may modulate gene transcription by affecting the stability, function, and activity of many regulators through a HSP90 phosphorylation-mediated chaperoning process. This study, using a quantitative, multidimensional separation phosphoproteomic approach that required a relatively low amount of cells, provides new insights into the diversity, variability, and dynamic nature of the protein phosphorylation/ dephosphorylation elicited by E2.