Ovarian cancer is the most fatal gynecological malignancy in women and identification of new therapeutic targets is essential for therapy of ovarian cancer. TRRAP (transformation/transcription domain-associated protein) is an adaptor protein and a component of histone acetyltransferase complex. The present study was undertaken to investigate the roles played by TRRAP in the proliferation and tumorigenicity of ovarian cancer stem cells. TRRAP expression was found to be up-regulated in sphere cultures of A2780 ovarian cancer cells. Knockdown of TRRAP significantly decreased cell proliferation and the number of A2780 spheroids. In addition, TRRAP knockdown induced cell cycle arrest and increased percentages of apoptotic A2780 cells. Notably, the mRNA levels of stemness-associated markers, that is, OCT4, SOX2, and NANOG, were suppressed in TRRAP-silenced A2780 sphere cells. In addition, TRRAP overexpression increased the mRNA level of NANOG and the transcriptional activity of NANOG promoter in these cells. Furthermore, TRRAP knockdown significantly reduced tumor growth in a murine xenograft transplantation model. Taken together, the present study suggests TRRAP plays an important role in the regulation of proliferation and stemness regulation of ovarian cancer stem cells.