Aggregation of Tau protein into neurofibrillary tangles is associated with the pathogenesis of Alzheimer’s disease (AD) which has no cure yet. Clearing neurofibrillary tangles is one of major therapeutic strategies. Experimental studies reported that norepinephrine (NE) has the ability to disrupt Tau filament and cause Tau degradation. However, the underlying mechanism remains elusive. Herein, we performed molecular dynamic simulations to investigate the influence of NE on the C-shaped Tau R3-R4 protofilament. Our simulations show that NE compound destabilizes Tau protofilament by mostly disrupting β6/β8 and altering the β2-β3 and β6-β7 angles. NE binds mainly with aromatic residues Y310/P312/H374/F378 through ππ stacking and charged residues E338/E342/D348/D358/E372 via hydrogen-bonding interactions. Our results, together with the findings that exercise can markedly increase NE level, suggest that exercise might be a potent therapy against AD. This study reveals the disruptive mechanism of Tau protofilament by NE molecules, which may provide new clues for AD drug candidate design.