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Estrogen receptors can regulate growth in papillary thyroid cancer and may affect prognosis after menopause. This study examines changes of estrogen receptor subtype ratio expression in papillary thyroid cancer cell lines derived from pre- and postmenopausal women.Cells were harvested from papillary thyroid cancer and non-papillary thyroid cancer thyroid tissue (control) from pre- (n = 9) and postmenopausal women (n = 11). Protein expression of estrogen receptor α, estrogen receptor β, and phosphorylated extracellular signal-regulated kinase and protein kinase B were analyzed. Matrix metalloproteinase-2 activity was determined as a measure of tumor invasiveness. Mitochondrial retrograde signaling was altered with ethidium bromide to determine its effect on estrogen receptor α protein expression.Estrogen receptor α expression was increased in postmenopausal papillary thyroid cancer cells compared with controls but was unchanged in premenopausal papillary thyroid cancer. Estrogen receptor β expression did not change in either group. Increased matrix metalloproteinase-2 activity was observed only in postmenopausal papillary thyroid cancer. Premenopausal papillary thyroid cancer cells demonstrated increased extracellular signal-regulated kinase and unchanged protein kinase B activation. Conversely, postmenopausal papillary thyroid cancer cells had decreased extracellular signal-regulated kinase and increased protein kinase B activation. Ethidium bromide treatment resulted in increased estrogen receptor α protein expression only in premenopausal papillary thyroid cancer cells.Increased estrogen receptor α expression may be involved in papillary thyroid cancer aggressiveness after menopause. This process may be regulated by differential activation of intracellular pathways and differing sensitivities to mitochondrial signaling regulation.