Renal cell carcinoma (RCC) originates in the lining of the proximal convoluted tubule and accounts for approximately 3% of adult malignancies. The RCC incidence rate increases annually and is twofold higher in males than in females. Female hormones such as estrogen may play important roles during RCC carcinogenesis and result in significantly different incidence rates between males and females. In this study, we found that estrogen receptor β (ERβ) was more highly expressed in RCC cell lines (A498, RCC-1, 786-O, ACHN, and Caki-1) than in breast cancer cell lines (MCF-7 and HBL-100); however, no androgen receptor (AR) or estrogen receptor α (ERα) could be detected by western blot. In addition, proliferation of RCC cell lines was significantly decreased after estrogen (17-β-estradiol, E2) treatment. Since ERβ had been documented to be a potential tumor suppressor gene, we hypothesized that estrogen activates ERβ tumor suppressive function, which leads to different RCC incidence rates between males and females. We found that estrogen treatment inhibited cell proliferation, migration, invasion, and increased apoptosis of 786-O (high endogenous ERβ), and ERβ siRNA-induced silencing attenuated the estrogen-induced effects. Otherwise, ectopic ERβ expression in A498 (low endogenous ERβ) increased estrogen sensitivity and thus inhibited cell proliferation, migration, invasion, and increased apoptosis. Analysis of the molecular mechanisms revealed that estrogen-activated ERβ not only remarkably reduced growth hormone downstream signaling activation of the AKT, ERK, and JAK signaling pathways but also increased apoptotic cascade activation. In conclusion, this study found that estrogen-activated ERβ acts as a tumor suppressor. It may explain the different RCC incidence rates between males and females. Furthermore, it implies that ERβ may be a useful prognostic marker for RCC progression and a novel developmental direction for RCC treatment improvement.