Apoptosis‐inducing factor (AIF) is a ubiquitous FAD‐binding flavoprotein comprised of 613 amino acids and plays an important role in caspase‐independent apoptosis. During apoptotic induction, AIF is translocated from the mitochondrial intermembrane space to the nucleus, where it interacts with DNA and activates a nuclear endonuclease. By performing a yeast two‐hybrid screen with mature AIF, we have isolated the eukaryotic translation initiation factor 3 subunit p44 (eIF3g). Our deletion mutant analysis revealed that the eIF3g N‐terminus interacts with the C‐terminal region of AIF. The direct interaction between AIF and eIF3g was confirmed in a GST pull‐down assay and also verified by the results of co‐immunoprecipitation and confocal microscopy studies. Using an in vitro TNT coupled transcription–translation system, we found that mature AIF could inhibit newly‐translated protein synthesis and this inhibition was significantly blocked by eIF3g competitively. These results were also confirmed in cells. In addition, mature AIF overexpression specifically resulted in the activation of caspase‐7, thereby amplifying the inhibition of protein synthesis including eIF3g cleavage. Our data suggest that eIF3g is one of the cytosolic targets that interacts with mature AIF, and provide insight into the AIF's cellular functions of the inhibition of protein synthesis during apoptosis.