Influenza viruses are respiratory pathogens that cause both seasonal and unpredictable pandemic infections in humans. Currently approved anti-influenza drugs target the viral proteins. A recurrent mutation in the influenza genome leads to drug resistance and thus hinders the efficacy of anti-influenza drug treatments. Influenza A virus (IAV) depends on host factors to complete its virus life cycle. Thus, there are increasing interests in antiviral drugs that target host cellular proteins required for virus replication. Poly (ADP-ribose) polymerases (PARPs) are the host factors that modify protein functions by adding ADP-ribose to target proteins. Using the CRISPR activation system, we screened all 17 PARP members for its effects on IAV infection in lung epithelial A549 cells. Tankyrase 1 and 2 (TNKS 1/2 or PARP5A/5B) were found to be potent proviral factors. Knockout of TNKS1 or TNKS2 in HEK293T cells by CRISPR reduced viral mRNA and protein levels in cells and viral titers in culture media. Double knockout of TNKS1 and TNKS2 had a larger effect on IAV infection than single knockout of each isoform. The effect of TNKS double knockout on IAV replication was strain independent. Overexpression of TNKS1/2 in the double knockout cells restored the IAV replication to a level similar to the control cells. TNKS double knockout activated JNK/c-Jun signaling, enhanced Stat signaling, and increased type I interferon expression. Finally, Tnks1 or Tnks2 KO mice challenged with a sublethal IAV showed increased type I IFN response and reduced viral load in the lungs. The survival rate of Tnks1 or Tnks2 KO mice from a lethal IAV infection was significantly increased compared to wild-type mice. In conclusion, TNKS1 and TNKS2 regulate influenza virus infection via type I interferon response.IMPORTANCEPoly (ADP-ribose) polymerases (PARPs) play a crucial role in DNA repair, cellular stress responses, epigenetics, gene transcription, and viral infections. However, the specific roles of PARPs in influenza A virus (IAV) infection remain unclear. In this study, we identified Tankyrase 1 and 2 (TNKS1/2 or PARP5a/5b) as the potent proviral factors. Knockout of TNKS1 or TNKS2 reduced viral replication in vitro, with the double knockout showing an even greater effect. TNKS double knockout also resulted in an increased type I interferon response to IAV infection. In vivo, Tnks1 or Tnks2 KO mice exhibited lower viral loads and higher survival rates following IAV challenge. Our findings highlight TNKS1/2 as important regulators of IAV infection and potential targets for antiviral therapies.