Purpose: How the neddylation pathway functions in breast tumor and regulation of estrogen receptor (ER) expression is rarely reported. The purpose of this study was to identify the role of neddylation in breast cancer and ER expression, and further explore the underlying mechanisms. Experimental Design: Expression patterns of nedd8- activating enzyme (NAE) and nedd8, two key proteins in the neddylation pathway, were examined in human breast specimens. ER-a expression was investigated using animal 18F-FES-PET/CT and immunoblotting upon NAE inhibitor MLN4924 treatment. Chromatin immunoprecipitation assay, luciferase reporter promoter assay, and the CRISPR-Cas9 system were used to elucidate the mechanism of ER-a regulation by MLN4924. The ER-positive breast cancer mouse model was used to determine the synergetic effect of MLN4924 and fulvestrant on tumor growth. All statistical tests were two-sided. Results: Both NAE1 and nedd8 expressions were higher in the ER-positive subgroup. Higher expressions of NAE1 and nedd8 indicated poorer prognosis. Importantly, ER-a expression was significantly downregulated upon MLN4924 treatment in vitro and in vivo. Mechanistically, MLN4924 treatment delayed serum and glucocorticoid-induced protein kinase (SGK) degradation and induced Forkhead box O3a (FOXO3a) nuclear export as well as decreased binding to the ESR1 promoter. Importantly, MLN4924 single or synergized with fulvestrant significantly suppressed the growth of ER-positive breast cancer in vitro and in vivo. Conclusions: Our proof-of-principle study determines the activation of neddylation in breast tumor tissues for the first time and reveals a new ER-a regulatory mechanism, as well as further explores an effective approach to improve fulvestrant sensitivity through a neddylation inactivation combination.