The Chinese herb berberine has versatile health effects. Recent reports indicate that berberine has the potential to prevent and treat Alzheimer's disease (AD). In the present study, we employed tau-expressing HEK293 cells (HEK293/tau) treated with calyculin-A as a cellular model to investigate the roles of berberine in cell viability, tau phosphorylation, and oxidative stress. We found a significant reduction of calyculin A-induced tau hyperphosphorylation at Ser198/199/202, Ser396, Ser404, Thr205, and Thr231 24 h after treatment with 20 μg/ml berberine. Berberine also restored protein phosphates 2A activity and reversed glycogen synthase kinase-3β (GSK-3β) activation, as determined by phosphatase activity assay and GSK-3β phosphorylation at Tyr216 and Ser9, respectively. Furthermore, berberine reversed both the increase of malondialdehyde and the decrease of superoxide dismutase activity induced by calyculin A, indicating its role in anti-oxidative stress. Our findings suggest that berberine may be a potential therapeutic drug for AD.