Endocrine therapy has become the most important treatment option for women with estrogen receptor (ER)-positive breast cancer. Urgently needed are prognostic assays that can identify those who need additional adjuvant therapy, such as signal transduction inhibitors or chemotherapy, for ER-positive early breast cancer. We examined phosphorylation of ERalpha serine (Ser) 118, ERalpha Ser167, p44/42 mitogen-activated protein kinase (MAPK), and Akt and expression of progesterone receptor, amplified in breast cancer 1 (AIB1), human epidermal growth factor receptor 2 (HER2), p53, and Ki67 in ER-positive breast cancers by immunohistochemistry, and analyzed their significance for prognosis. Phosphorylation levels of ERalpha Ser118, ERalpha Ser167, MAPK, and Akt were positively correlated. AIB1 expression was significantly associated with phosphorylation of ERalpha Ser118, MAPK, and Akt, and HER2 expression. Low phosphorylation of ERalpha Ser118 and high phosphorylation of ERalpha Ser167 were associated with significantly improved disease-free (P=0.0003 and P=0.0002 respectively) and overall survival (P=0.0007 and P=0.0016 respectively) in multivariate analyses. Our data suggest that phosphorylation of ERalpha Ser118 and ERalpha Ser167 affects survival in ER-positive breast cancer and could be helpful in distinguishing patients who are likely to benefit from endocrine therapy alone from those who are not.