Earlier, we proposed that the ability of folic acid and vitamin B(12) to preserve systemic and mitochondrial function after short-term exposure to arsenic may prevent further progression to more permanent injury and pathological changes leading to cell death. To elucidate its mechanism, the present study examined the antiapoptotic efficacy of folic acid and vitamin B(12) against short-term arsenic exposure-induced hepatic mitochondria oxidative stress and dysfunction. Sixteen to eighteen weeks old male albino rats weighing 140-150 × g were divided into five groups: Control (A), Arsenic-treated (B), Arsenic + folic acid (C), Arsenic +vitamin B(12) (D), and Arsenic + folic acid + vitamin B(12) (E). Data generated indicated that folic acid and vitamin B(12) separately or in combination can give significant protection against alterations in oxidative stress and apoptotic marker parameters and downstream changes in mitochondria, namely pro-oxidative (NO, TBARS, OH(-)) and antioxidative defense (SOD, CAT, GSH) markers, iNOS protein expression, mitochondrial swelling, cytochrome c oxidase and Ca(2+)-ATPase activity, Ca(2+) content, caspase-3 activity. Additionally, results of hepatic cell DNA fragmentation, arsenic load of blood, hepatic tissue and urine, and histological observations, all strongly support that both these supplements have efficacy in preventing apoptotic changes and cellular damage. As the mechanisms of actions of both of these supplements are methylation related, a combined application was more effective. Results further reveal new molecular targets through which folic acid and vitamin B(12) separately or in combination work to alleviate one critical component of arsenic-induced liver injury: mitochondria dysfunction. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2010.