Avian influenza A viruses generally do not replicate efficiently in human cells， but substitution of glutamic acid (Glu， E) for lysine (Lys， K) at residue 627 of avian influenza virus polymerase basic protein 2 (PB2) can serve to overcome host restriction and facilitate human infectivity. Although PB2 residue 627 is regarded as a species-specific signature of influenza A viruses， host restriction factors associated with PB2E have yet to be fully investigated. We conducted immunoprecipitation， followed by differential proteomic analysis， to identify proteins associating with PB2K (human signature) and PB2E (avian signature) of influenza A/WSN/1933(H1N1) virus， and the results indicated that Tu elongation factor， mitochondrial (TUFM)， had a higher binding affinity for PB2E than PB2K in transfected human cells. Stronger binding of TUFM to avian-signature PB2G/Q and PB2E in the 2009 swine-origin pandemic H1N1 and 2013 avian-origin H7N9 influenza A viruses was similarly observed. Viruses carrying avian-signature PB2E demonstrated increased replication in TUFM-deficient cells， but viral replication decreased in cells overexpressing TUFM. Interestingly， the presence of TUFM specifically inhibited the replication of PB2E viruses， but not PB2K viruses. In addition， enhanced levels of interaction between TUFM and PB2E were noted in the mitochondrial fraction of infected cells. Furthermore， TUFM-dependent autophagy was reduced in TUFM-deficient cells infected with PB2E virus; however， autophagy remained consistent in PB2K virus-infected cells. The results suggest that TUFM acts as a host restriction factor that impedes avian-signature influenza A virus replication in human cells in a manner that correlates with autophagy. An understanding of the mechanisms that influenza A viruses utilize to shift host tropism and the identification of host restriction factors that can limit infection are both critical to the prevention and control of emerging viruses that cross species barriers to target new hosts. Using a proteomic approach， we revealed a novel role for TUFM as a host restriction factor that exerts an inhibitory effect on avian-signature PB2E influenza virus propagation in human cells. We further found that increased TUFM-dependent autophagy correlates with the inhibitory effect on avian-signature influenza virus replication and may serve as a key intrinsic mechanism to restrict avian influenza virus infection in humans. These findings provide new insight regarding the TUFM mitochondrial protein and may have important implications for the development of novel antiviral strategies.