Virus-host interactions are important determinants of virus replication and immune responses, but they are not well-defined. In this study we analyzed quantitative host protein alterations in nuclei-enriched fractions from multiple primary human bronchial airway epithelial (HBAE) cells infected by an H1N1 influenza A virus (A/PR/8/34). We first developed an effective detergent-free nuclear lysis method that was coupled with in-solution digestion and LC-MS/MS. Using SILAC, we identified 837 HBAE nuclear proteins in three different donors and compared their responses to infection at 24 h. Some proteins were altered in all three donors, of which 94 were up-regulated and 13 were down-regulated by at least 1.5-fold. Many of these up-regulated proteins clustered into purine biosynthesis, carbohydrate metabolism, and protein modification. Down-regulated proteins were not associated with any specific pathways or processes. These findings further our understanding of cellular processes that are altered in response to influenza in primary epithelial cells and may be beneficial in the search for host proteins that may be targeted for antiviral therapy.