Latent genetic variations of cholesterol metabolism-related genes in late-onset Alzheimer’s disease, especially, as well as in mild cognitive impairment pathogenesis are still to be studied extensively. Thus, we performed the targeted-sequencing of 12 nuclear receptor genes plus APOE which were involved in cholesterol content modulation to screen susceptible genetic variants and focused on a new risk variant ESR1 rs9340803 at 6q25.1 for both late-onset Alzheimer’s disease (OR=3.30[1.84~4.22], p<0.001) and mild cognitive impairment (OR=3.08[1.75~3.89], p<0.001). This low-frequency variant was validated in three independent cohorts totaling 854 late-onset Alzheimer’s disease cases, 1059 mild cognitive impairment cases and 1254 controls from nine provinces of China mainland. Preliminary functional study on it revealed decreased ESR1 expression in vitro. Besides, we detected higher serum Aβ1-40 concentration in participants carrying this variant (p=0.038) and lower plasma total cholesterol level in this variant carriers with late-onset Alzheimer’s disease (p=0.009). In summary, we identified a susceptible variant which might contribute to developing mild cognitive impairment at earlier stage and Alzheimer’s Disease later. Our study would provide new insight into the disease causation of late-onset Alzheimer’s disease and could be exploited therapeutically.