The 40 S ribosomal S6 kinase 1 (S6K1) acts downstream of mTOR (mammalian target of rapamycin) and is sensitive to inhibition by rapamycin. The chromosomal region 17q23 containing the RPS6KB1 gene is frequently amplified in breast cancer cells, leading to S6K1 overexpression. The role of S6K1 in disease development and progression is supported by the observation that S6K1 overexpression is associated with poor prognosis in breast cancer patients. However, the identity of mammary cell-specific S6K1 targets is not well understood. In this study, we report that overexpression of S6K1 endows breast cancer cells with a proliferative advantage in low serum conditions and enhanced sensitivity to rapamycin. We investigate the molecular mechanism behind this observation to show that S6K1 regulates estrogen receptor alpha (ERalpha) by phosphorylating it on serine 167, leading to transcriptional activation of ERalpha. By contributing to the activation of ERalpha, S6K1 promotes ERalpha-mediated cell proliferation and may be a target of therapeutic intervention in breast cancer.