Parkinson's disease is a debilitating neurodegenerative disease characterized by loss of midbrain dopaminergic neurons. These neurons are particularly sensitive to the neurotoxin 1‐methyl‐4‐phenylpyridinium (MPP⁺), the active metabolite of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), which causes parkinsonian syndromes in humans, monkeys and rodents. Although apoptotic cell death has been implicated in MPTP/MPP⁺ toxicity, several recent studies have challenged the role of caspase‐dependent apoptosis in dopaminergic neurons. Using the midbrain‐derived MN9D dopaminergic cell line, we found that MPP⁺ treatment resulted in an active form of cell death that could not be prevented by caspase inhibitors or over‐expression of a dominant negative inhibitor of apoptotic protease activating factor 1/caspase‐9. Apoptosis inducing factor (AIF) is a mitochondrial protein that may mediate caspase‐independent forms of regulated cell death following its translocation to the nucleus. We found that MPP⁺ treatment elicited nuclear translocation of AIF accompanied by large‐scale DNA fragmentation. To establish the role of AIF in MPP⁺ toxicity, we constructed a DNA vector encoding a short hairpin sequence targeted against AIF. Reduction of AIF expression by RNA interference inhibited large‐scale DNA fragmentation and conferred significant protection against MPP⁺ toxicity. Studies of primary mouse midbrain cultures further supported a role for AIF in caspase‐independent cell death in MPP⁺‐treated dopaminergic neurons.