Breast cancer is the most diagnosed cancers among American women. The oxidative stress from an imbalance between reactive oxidants and antioxidants is associated with carcinogenesis and is an important risk factor for breast cancer. Nrf2, a transcription factor, responds to oxidative and electrophilic stress. However, Nrf2 plays dual roles in carcinogenesis: in normal and premalignant cells, activation of Nrf2 would weaken further oxidation and prevent carcinogenesis; in malignant cells, overexpression of Nrf2 could protect tumor cells from cytotoxic effects of elevated endogenous reactive oxygen species. The beneficial or detrimental effects of Nrf2 also depends on environmental conditions. Ursolic Acid (UA), a natural pentacyclic triterpenoid widely distributed in fruits, herbs and spices, has been reported to have a strong anti-proliferative activity towards human breast cancer cells including malignant MDA-MB-231 human breast cancer cells. However, it is not known whether UA has effects on Nrf2 in MDA-MB-231 human breast cancer cells. In this study, UA inhibited the expression of Nrf2 and its phosphorylated form (phosphor S40) p-Nrf2 in MDA-MB-231 human breast cancer cells in whole cells and nucleus. The elevation in NQO1, the Nrf2 downstream antioxidant enzymes, and, the reduction in Keap1, the Nrf2 repressor, were also observed. After a 12 h pretreatment with EGF, the p-EGFR and total cellular and nuclear p-Nrf2 in MDA-MB-231 human breast cancer cells were induced but no significant differences were observed in total cellular and nuclear Nrf2 expressions. It may indicate EGFR phosphorylation might be an upstream for p-Nrf2. Tamoxifen, a selective ER modulator approved drug in the US to reduce breast cancer risk, has been proved to be an effective treatment in metastatic breast cancer and the tamoxifen adjuvant therapy reduced breast cancer recurrence and death. However, the tamoxifen resistance is a critical challenge for the treatment. We compared the constitutive expressions of Nrf2 in ER positive MCF-7 human breast cancer cells and tamoxifen-resistant MCF-7 cells (MCF-7TamR). Nrf2 and p-Nrf2 were overexpressed in MCF-7TamR cells. Its downstream proteins SOD1 and NQO1 in MCF-7TamR cells was distinctly higher in MCF-7TamR cells than in parental MCF-7 cells. The expression level of Keap1, the cytosolic repressor of Nrf2, was much lower in MCF-7TamR cells. The results suggested that Nrf2/ARE pathway might be therapeutic target to overcome tamoxifen resistance in human breast cancers. UA significantly inhibited the expression of Nrf2 and its phosphorylated form (phosphor S40) p-Nrf2 in MCF-7 and MCF-7TamR human breast cancer cells. The inhibitory effects of UA on NQO1 and SOD1 were also shown in cells. A stronger anti-proliferative activity against MCF-7TamR cells than parental MCF-7 cells at the concentration of 9 µM and higher was shown. The significant differences in Nrf2 inhibition by UA between MCF-7 and MCF-7TamR cells were also observed. It suggested that the anti-proliferative effect of UA might be accompanied with Nrf2 inhibition in MCF-7 and MCF-7TamR human breast cancer cells according to our findings. Furthermore, the combination of UA plus tamoxifen was demonstrated a synergistic anti-proliferative effect in MCF-7 human breast cancer cells, which may suggest that the combined approach of UA and tamoxifen may have greater therapeutic effect on breast cancers than individual compound.