The PB2-E627K mutation is a key determinant for mammalian adaptation of avian influenza A viruses, yet its system-wide impact on the host microenvironment of the viral polymerase remains unknown. Here, we employed TurboID proximity labeling coupled with quantitative mass spectrometry to map the proximal interactomes of the reconstituted H5N1 polymerase trimer (PA-PB1-PB2) in its wild-type (WT) and PB2-E627K forms in human cells. We identified 204 high-confidence interactions and revealed that E627K extensively rewires the host interaction landscape, gaining associations with cytoskeletal regulators while losing connections to mRNA splicing machinery. AlphaFold3 structural modeling provided high-confidence predictions for key interactions, including PB2-KPNA1 and PA-CPSF6, which were validated biochemically. This study provides the first spatial interactome map of a pivotal influenza host-adaptation mutation, demonstrating how PB2-E627K reprogrammes the host-virus interface to facilitate cross-species infection, and offers a resource for novel antiviral strategies.