It is recognized that influenza virus induces caspase-dependent apoptosis by activating caspase-3. Apoptosis-inducing factor (AIF) is a caspase-independent cell death effector, and its mitochondrial-nuclear translocation plays an important role in apoptosis. It is demonstrated in this study how influenza virus infection can induce caspase-independent apoptosis in the human alveolar epithelial cell line A549. AIF is translocated from the mitochondria to the nucleus in a caspase-independent manner in response to infection with influenza virus. Knockdown of AIF expression by small interfering RNA (siRNA) led to a reduction in virus-infection-induced apoptosis and virus yield. These results indicate that AIF translocation has a role in influenza-virus-induced apoptosis.