Swine flu caused by swine influenza A virus (swIAV) is an acute respiratory viral disease that is spreading in swine herds worldwide. Although the effect of some host factors on replication of swIAV has been identified, the role of CD46 in this process is unclear. Here, we report that CD46 inhibits the replication of swIAV by promoting the production of type I interferons (IFNs) in porcine kidney (PK-15) cells. CD46 knockout (CD46-KO) and stably expressing (CD46-overexpression) PK-15 cells were prepared using lentivirus-mediated CRISPR/Cas9 gene editing and seamless cloning technology. The results of virus infection in CD46-overexpression PK-15 cells showed that the replication of H1N1 and H3N2 swIAVs were inhibited, and the production of type I IFNs (IFN-α, IFN-β), interferon regulatory factor (IRF) 3, and mitochondrial antiviral-signaling protein (MAVS) was enhanced. Virus infection in CD46-KO PK-15 cells showed the opposite results. Further results showed that CD46-KO PK-15 cells have a favorable ability to proliferate influenza viruses compared to Madin-Darby canine kidney (MDCK) and PK-15 cells. These findings indicate that CD46 acts as promising target regulating the replication of swIAV, and help to develop new agents against infection and replication of the virus.