The estrogen receptor (ER) plays an important role in breast cancer development and progression. Hypoxia modulates the level of ERα expression and induces ligand-independent transcriptional activation of ERα, which is closely related with the biology of breast carcinomas. Since phosphorylation itself affects the transcriptional activity and stabilization of ERα, we examined changes in ERα phosphorylation under hypoxic conditions. Hypoxia induced phosphorylation of ERα at serine residue 118 (S118) in the absence of estrogen through the mitogen-activated protein kinase (MAPK)/ERK1/2 pathway. Cell proliferation was significantly decreased under normoxia or hypoxia when ERα harboring the S118A mutation was overexpressed. Our previous studies showed that ER degradation is the most prominent phenomenon under hypoxia. E2-induced ER protein downregulation is dependent on phosphorylation of S118. However, hypoxia-induced ERα degradation did not involve S118 phosphorylation. Our study implies the existence of a differential mechanism between E2 and hypoxia-mediated ERα protein degradation. Understanding the mechanistic behavior of ER under hypoxia will likely facilitate understanding of endocrine therapy resistance and development of treatment strategies for breast cancer.