Mitochondrial dysfunction plays a fundamental role in the pathogenesis of cognitive deficit. Rutaecarpine (Rut) is a natural alkaloid with anti-inflammatory and antioxidant properties. This study explored whether Rut treatment could enhance cognitive function by improving mitochondrial function and examined the potential mechanisms underlying this ameliorative effect. We used the Morris water maze and Y-maze tests to evaluate the behavioral effects of Rut in a mouse model of cognitive impairment induced by subcutaneous injection of D-galactose (D-gal). Furthermore, we assessed the effects of Rut on mitochondrial function using cell viability assays, flow cytometry, western blotting, biochemical analysis, and immunochemical techniques in vivo and in vitro. The results indicated Rut treatment attenuated cognitive deficits and mitochondrial dysfunction in the mouse model. Similarly, it maintained the balance of mitochondrial dynamics in neurocytes and reduced oxidative stress and mitochondrial apoptosis in the HT22 cell model. Moreover, we found that these protective effects were dependent on the activation of the AMP-activated protein kinase/proliferator-activated receptor gamma coactivator 1-alpha (AMPK/PGC1 & alpha;) signaling pathway. Our data indicate that Rut treatment are sensitive to reversal cognitive deficits and mitochondrial dysfunction induced by D-gal; this suggests that Rut is a promising mitochondria-targeted therapeutic agent for treating cognitive impairment.