Overdose of acetaminophen (APAP) is a common cause of acute liver failure. Oxidative stress and mitochondrial dysfunction are related to APAP-induced hepatotoxicity. This study investigated the protective role of betaine as a methyl donor and S-adenosylmethionine precursor against APAP hepatotoxicity in isolated mice liver mitochondria. We treated male NMRC mice with 125, 250, 500, and 1000 mg/kg betaine for 5 days followed by 300 mg/kg APAP 1 h later. At 24 h after APAP administration, animals were sacrificed and liver mitochondria were isolated by different centrifugation methods. Biochemical, histological and immunohistochemical analyses were then conducted. Pretreatment of mice with 250, 500, and 1000 mg/kg betaine ameliorated hepatotoxicity (serum ALT and AST activity and histopathology finding); reversed mitochondrial complex II activity, glutathione level, superoxide dismutase, glutathione peroxidase and catalase activity, glutamate cysteine ligase catalytic protein expression, and mitochondrial membrane potential; and suppressed mitochondrial lipid peroxidation and reactive oxygen species. A contradictory effect was observed in mice treated with APAP. Betaine (500 mg/kg) was chosen as the most effective dose to rescue APAP hepatotoxicity in mice. These findings confirmed that betaine plays a protective role against APAP hepatotoxicity via protecting mitochondrial complex II and regenerating mitochondrial GSH levels by increasing GCLC expression. Betaine displayed antioxidant actions different from other antioxidants via modifying cysteine supply in the transsulfuration pathway in the liver. These findings suggested the potential of betaine as an alternative agent for the treatment of hepatotoxicity induced by APAP.