Apoptosis-inducing factor (AIF), normally confined to mitochondria, can move from mitochondria to nuclei, participate in the execution of cell death. The mechanism by which AIF induces cell apoptosis is unclear. In order to analyze proteins which may interact with AIF, interceptive AIF gene (mitochondrial localization sequence, MLS, was deleted) which was amplified from the total mRNA extracted from hepatoma HepG2 cells by reverse transcriptase polymerase chain reaction(RT-PCR), was inserted into pcDNA3.1 plasmid.The recombinant plasmids constructed successfully were then transfected into HepG2 cells and expressions of interceptive AIF were detected by RT-PCR and Western blot. Immunoprecipitation (IP) and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were used to isolate and identify proteins which can interact with interceptive AIF respectively. Screened out proteins which may interact with AIF were then confirmed by CO-IP and Western blot. The result showed that AIF may interact with β-actin.