The human RNA polymerase II (RNAPII)-associated factor complex (hPAFc) and its individual subunits have been implicated in human diseases, including cancer. However, its involvement in breast cancer awaits investigation. Using data mining and human breast cancer tissue microarrays, we found that Ctr9, the key scaffold subunit in hPAFc, is highly expressed in estrogen receptor α-positive (ERα+) luminal breast cancer, and the high expression of Ctr9 correlates with poor prognosis. Knockdown of Ctr9 in ERα+ breast cancer cells almost completely erased estrogen-regulated transcriptional response. At the molecular level, Ctr9 enhances ERα protein stability, promotes recruitment of ERα and RNAPII, and stimulates transcription elongation and transcription-coupled histone modifications. Knockdown of Ctr9, but not other hPAFc subunits, alters the morphology, proliferative capacity, and tamoxifen sensitivity of ERα+ breast cancer cells. Together, our study reveals that Ctr9, a key subunit of hPAFc, is a central regulator of estrogen signaling that drives ERα+ breast tumorigenesis, rendering it a potential target for the treatment of ERα+ breast cancer.