Background and Purpose:: Peroxisome proliferator-activated receptor α and-γ (PPARα/γ) are known to play crucial roles in acute liver injury (ALI). Icariside Ⅱ (ICS Ⅱ), a natural flavonoid compound derived from Herba Epimedii, confers neuroprotection with PPARα/γ induction potency. This study was aimed to explore whether ICS Ⅱ has the capacity to protect against ALI, and if so what are the role of PPARα/γ in the beneficial effect of ICS Ⅱ on ALI. Experimental Approach: Mice challenged by D-galactosamine (GalN)/lipopolysaccharide (LPS) and Kupffer cells (KCs) upon LPS insult were used as ALI models in vivo and in vitro. PPARα/γ-deficient mice and Sirt6-deficient mice were treated with ICS Ⅱ to validate the potential targets of ICS Ⅱ on ALI. Key results: ICS Ⅱ dose-dependently improved the survival rate and liver histology, decreased ALT and AST in D-galactosamine (GalN)/lipopolysaccharide (LPS)-treated mice. Furthermore, ICS Ⅱ directly bound to PPARα/γ and increased their activities. The protective properties of ICS Ⅱ were counteracted when PPARα/γ were knocked out in GalN/LPS-induced mice and LPS-induced KCs, respectively. Mechanistically, ICS Ⅱ restored mitochondrial function, reduced oxidative stress and inflammation through activating PPARα/γ, which interacted with Sirt6 and inhibited NF-κB nuclear translocation. Intriguingly, ICS Ⅱ-evoked hepatoprotective effect and activation of PPARα/γ were largely blunted in Sirt6-deficient mice. Conclusions and implications: Our findings not only highlight PPARα/γ-SIRT6 signaling as a vital therapeutic target to combat ALI, but also reveal ICS Ⅱ may serve as a novel dual PPARα/γ agonist to safeguard ALI from the oxidation-inflammation vicious circle by coactivating SIRT6.