Updated project metadata. Human respiratory syncytial virus (RSV) is the leading cause of severe acute lower respiratory tract infections in infants worldwide. Non-structural protein NS1 of RSV modulates the host innate immune response by acting as an antagonist of type I and type III interferon (IFN) production and signaling in multiple ways. It is likely that NS1 performs this function by interacting with different host proteins. In order to obtain a comprehensive overview of NS1 interaction partners, we performed three complementary protein-protein interaction screens i.e. BioID, MAPPIT and KISS. The BioID proximity screen was performed during an RSV infection in A549 cells. MED25, a subunit of the Mediator complex, was identified in all 3 performed screening methods as a potential NS1 interacting protein. We confirmed the interaction between MED25 and RSV NS1 by co-immunoprecipitation, not only upon overexpression of NS1, but also with endogenous NS1 during RSV infection. We also demonstrate that the replication of RSV is enhanced in MED25 knockout A549 cells, suggesting a potential antiviral role of MED25 during RSV infection. Mediator subunits function as transcriptional coactivators and are involved in transcriptional regulation of their target genes. Therefore, the interaction between RSV NS1 and cellular MED25 might be beneficial during an RSV infection as this can affect host transcription and the host immune response to infection.