Several reports indicate crosstalk between the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and estrogen, which has a protective effect in colorectal cancer (CRC). The aim of this study was to investigate the role of Nrf2 signaling in the anti-inflammatory effect of estrogen using Nrf2 knockout (Nrf2 KO) mouse embryonic fibroblasts (MEFs), a powerful system to test the function of target genes due to their easy accessibility, and rapid growth rates. After inducing inflammation by tumor necrosis factor alpha (TNF-α), the effects of 17β-estradiol (E2) on the expression of proinflammatory mediators [i.e., NF-κB and inducible nitric oxide synthase (iNOS)] and estrogen receptors were evaluated by Western blot. In wild type (WT) MEFs, E2 treatment ameliorated TNF-α-induced nuclear translocation of NF-κB and expression of its target protein iNOS. Estrogen receptor beta (ERβ) expression was decreased by TNF-α-induced inflammation and restored by E2 treatment. When treated to WT MEFs, E2 induced nuclear translocation of Nrf2. The inhibitory effect of E2 on TNF-α-induced enhancement of iNOS was markedly dampened in Nrf2 KO MEFs. Notably, ERβ expression was significantly diminished in Nrf2 KO MEFs compared to that in WT cells. Promoter Database (EPD) revealed two putative anti-oxidant response elements (AREs) within the mouse ERβ promoter. Furthermore, in WT MEFs, E2 treatment repressed TNF-α-induced expression of iNOS protein and recovered by 4-(2-phenyl-5,7-bis(trifluoromethyl)pyrazolo(1,5-a)pyrimidin-3-yl)phenol (PHTPP), a selective ERβ antagonist, treatment, but not in Nrf2 KO MEFs. In conclusion, Nrf2 plays a pivotal role in the anti-inflammatory of estrogen by direct regulating the expression of ERβ.