Influenza viruses (IVs) trigger a series of intracellular signaling events and induce complex cellular responses from the infected host cell. Accumulating evidence suggests that host cell proteins play an essential role in viral propagation and represent novel antiviral therapeutic targets. Subcellular proteomic technology provides a method for understanding regional differences at the protein level. The present study, which utilized subcellular proteomic technology, aimed to identify host cell proteins involved in influenza virus (HIN1) infection. Two-dimensional gel electrophoresis (2-DE) combined with mass spectrum (MS) was performed on protein extracts from the nuclei, cytoplasm, and mitochondria of infected and control human lung epithelial cells (A549). In total, 112 differentially expressed protein molecules were identified; 80 protein spots were successfully validated using MS. The differential expression of ISG15, MIF, PDCD5, and UCHL1 was confirmed by western blot. Furthermore, antisense oligodeoxyribonucleotide (ODN) targeting ISG15, MIF, PDCD5, and UCHL1 significantly mitigated HIN1 propagation, cytopathic effects, vRNA by RT-qPCR, and rescued cell viability in A549 cells. Taken together, the differentially expressed proteins identified in this study might provide novel targets for anti-influenza drug development.