Quantitative phosphoproteome and transcriptome analysis of ligand-stimulated MCF-7 human breast cancer cells was performed to understand the mechanisms of tamoxifen resistance at a systems level. Phosphoproteome data revealed that wild type (WT) cells were more enriched with phospho-proteins than tamoxifen-resistant (TamR) cells after stimulation with ligands. Surprisingly, decreased phosphorylation after ligand perturbation was more common than increased phosphorylation. In particular, 17beta-estradiol (E2) induced down-regulation in WT cells at a very high rate. E2 and the ErbB ligand, heregulin (HRG) induced almost equal numbers of up-regulated phospho-proteins in WT cells. Pathway and motif activity analyses using transcriptome data additionally suggested that deregulated activation of GSK3B(glycogen synthase kinase 3 beta) and MAPK1/3 signaling might be associated with altered activation of CREB and AP-1 transcription factors in TamR cells and this hypothesis was validated by reporter assays. An examination of clinical samples revealed that, inhibitory phosphorylation of GSK3B at serine 9 was significantly lower in tamoxifen-treated breast cancer patients that eventually had relapses, implying that activation of GSK3B may be associated with the tamoxifen resistant phenotype. Thus, the combined phosphoproteome and transcriptome dataset analyses revealed distinct signal-transcription programs in tumor cells and provided a novel molecular target to understand tamoxifen resistance.