Abstract Breast cancer is a hormone-related carcinoma and the most commonly diagnosed malignancy among women. Although human epidermal growth factor receptor-2 (Her2), estrogen receptor (ER), and progesterone receptor (PR) are the major diagnostic markers and therapeutic targets to breast cancer, searching for additional molecular targets becomes an important issue and one of the candidates is androgen receptor (AR). In recent studies, AR expresses in 70% breast cancer cases and its expression connects to low recurrence and high survival rate. Our previous study shows that S81 phosphorylation is critical for AR protein stability and modulated by Her2 in prostate cancer cells. In this study, we explore the roles of AR and Her2 in proliferation of breast cancer cell line, MDA-MB-453. The data show that AR which was activated by synthetic androgen R1881 decreased proliferation of MDA-MB-453 cells. AR knockdown increased both phosphorylation of Akt and ERK. Notably, AR activation decreased protein level of Early growth response 1 (Egr1) and increased protein level of cyclin-dependent kinase inhibitor p27, which suggests the correlation of AR activation and proliferation suppression. Besides, Her2 activation by Heregulin (HRG) decreased the levels of p-S81-AR. Her2 inhibition by small molecular inhibitor, Lapatinib, increased the levels of p-S81-AR and p27. In summary, our results indicate that Her2 might negatively regulate AR activation and contribute to suppressive proliferation of MDA-MB 453 cells. Citation Format: Pao-Hsuan Huang, Chen-Chuan Huang, Yueh-Tsung Lee, Chia-Herng Yue, Ho Lin. Her2 affects AR phosphorylation at serine 81 and suppresses cell proliferation of breast cancer cell line MDA-MB-453. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2123. doi:10.1158/1538-7445.AM2014-2123