Abstract Sequential proteolytic cleavage of amyloid precursor protein (APP) by β-secretase (BACE1) is a crucial process in β-amyloid peptide (Aβ) generation, which further forms into neurotoxic amyloid plaques that are considered to be a pivotal hallmark in the development and progress of Alzheimer's disease (AD). Hence, the inhibition of BACE1 has emerged as a credible target for the prevention and/or treatment of AD. In this study, γ-linolenic acid (GLA) was discovered as a novel BACE1 specific inhibitor. GLA non-competitively suppressed BACE1 activity with an IC50 value of 7.6 × 10−5 M and Ki value of 3.5 × 10−5 M. In addition, we demonstrated the calculated docking poses of GLA to human BACE1 and revealed the interactions of GLA with the allosteric site of the enzyme bound with the OH group of CYS359. Our findings provide a novel possibility of GLA to be efficacious for the prevention of AD and provide scaffolds to explore more potent natural BACE1 inhibitors.