The antioxidant effects alonenin, the main active ingredient of Aloe vera were investigated in vitro and in vivo. Aloenin increased the survival rate of oxidatively damaged cells and reduced cellular malondialdehyde production. It also increased cellular superoxide dismutase, catalase, and glutathione peroxidase (GPx) enzyme activities. An acute liver injury mouse model was constructed using CCl₄, and aloenin downregulated the liver index, enhanced the total antioxidant capacity of liver tissue, and alleviated oxidative stress injury. In addition, aloenin upregulated the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and glutamate-cysteine ligase catalytic subunit genes and proteins, and reduced the expression of kelch-like eCH-associated protein 1 (Keap1) genes and proteins in mouse liver tissues. Overall, aloenin showed protective effects against oxidative stress in HepG2 cells and liver injury in mice. This may be related to the activation of the Keap1/Nrf2/HO-1 and phosphatidylinositol 3-kinase/protein kinase B signaling pathways.