17β-estradiol (E2) is an important endocrine hormone underlying mammalian body, which participates in the regulatory of physiological functions of reproductive system, mammary gland, bone and cardiovascular, et.al. Paradoxically, despite the physiological actions of endogenous E2 (0.2-1.0 nmol/L), numerous clinical and experimental studies demonstrated that high-dose E2 treatment could cause tumor regression and exert pro-apoptotic actions in multiple cells, but the underlying mechanism is still undescribed. Especially, little information of cellular processes responding to lethality of 17β-estradiol is available. In the present work, we attempted to characterize the cellular processes responding to high-dose (mol/L) E2 treatment by quantitative phosphoproteomics to help understand the regulatory mechanism of E2 induced cell death. Therefore, to analyze the cell phenotype induced by high-dose (umol/L) E2, cell counting kit-8 assay (CCK8), cell cytotoxicity analysis by trypan blue staining and microscopy imaging were first performed on HeLa cells following 1-10 mol/L E2 or dimethyl sulfoxide (DMSO) treatment for 1-3 days. As a result, E2 was found to inhibit cell proliferation and induce cell death in a dose-dependent and time-dependent fashion. Specially, in comparison with DMSO-treated HeLa cell samples, 5 umol/L E2 treatment for 2 days resulted in > 74% of growth inhibition and about 50% of cell death, which was employed for following quantitative phosphoproteomic analysis. To investigate the intracellular cellular processes responding to high-dose (umol/L) E2 treatment, solid phase extraction (SPE)-based immobilized titanium ion affinity chromatography (Ti-IMAC) phosphopeptide enrichment method coupled with data independent acquisition (DIA)-based quantitative proteomics was employed for in-depth screening of high-dose E2 regulated phosphorylation sites. As a result, more than 10000 phosphorylation sites were identified from the E2 and DMSO treated HeLa cell samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Among these phosphorylation sites, in comparison with the DMSO-treated cell samples, 5 umol/L E2 treatment caused an increase of 537 phosphorylation sites on 372 proteins and a decrease of 387 phosphorylation sites on 256 proteins with a threshold of p-value < 0.01 and |fold change|≥2. Totally 924 phosphorylation sites on 599 proteins were significantly regulated by high-dose (umol/L) E2, which were then performed for enrichment analysis. In addition, 453 phosphorylation sites on 325 proteins were found to be identified in only E2 or DMSO treated cell samples. These phosphorylation sites probably be phosphorylated or de-phosphoylated responding to high-dose E2 stimulation, which likely be regulated by high-dose (umol/L) E2 and were also performed for enrichment analysis in parallel. Taken together, 1218 phosphorylation sites on 741 proteins were significantly regulated by high-dose E2.