Background: Estrogen receptor (ER) beta has been suggested to affect ovarian carcinogenesis. We examined the effects of four ER beta agonists on proliferation and gene expression of two ovarian cancer cell lines. Methods: OVCAR-3 and OAW-42 ovarian cancer cells were treated with the ER beta agonists ERB-041, WAY200070, Liquiritigenin and 3 beta-Adiol and cell growth was measured by means of the Cell Titer Blue Assay (Promega). ER beta expression was knocked down by transfection with specific siRNA. Additionally, transcriptome analyses were performed by means of Affymetrix GeneChip arrays. To confirm the results of DNA microarray analysis, Western blot experiments were performed. Results: All ER beta agonists tested significantly decreased proliferation of OVCAR-3 and OAW-42 cells at a concentration of 10 nM. Maximum antiproliferative effects were induced by flavonoid Liquiritigenin, which inhibited growth of OVCAR-3 cells by 31.2% after 5 days of treatment, and ERB-041 suppressing proliferation of the same cell line by 29.1%. In OAW-42 cells, maximum effects were observed after treatment with the ER beta agonist WAY200070, inhibiting cell growth by 26.8%, whereas ERB-041 decreased proliferation by 24.4%. In turn, knockdown of ER beta with specific siRNA increased cell growth of OAW-42 cells about 1.9-fold. Transcriptome analyses revealed a set of genes regulated by ER beta agonists including ND6, LCN1 and PTCH2, providing possible molecular mechanisms underlying the observed antiproliferative effects. Conclusion: In conclusion, the observed growth-inhibitory effects of all ER beta agonists on ovarian cancer cell lines in vitro encourage further studies to test their possible use in the clinical setting.