Using SPR, molecular docking, along with cellular and animal experiments, we confirmed that beta-site amyloid precursor protein cleaving enzyme (BACE1) and amyloid beta1-42 (Aβ1-42) are direct targets of berberine, elucidating its pharmacological basis in the treatment of Alzheimer’s disease. Here, BACE1 is linked to early-onset autosomal dominant Alzheimer disease.