The process of nucleocytoplasmic shuttling is mediated by karyopherins. Dysregulated expression of karyopherins may trigger oncogenesis through aberrant distribution of cargo proteins. Karyopherin subunit alpha-2 (KPNA2) was previously identified as a potential biomarker for nonsmall cell lung cancer by integration of the cancer cell secretome and tissue transcriptome data sets. Knockdown of KPNA2 suppressed the proliferation and migration abilities of lung cancer cells. However, the precise molecular mechanisms underlying KPNA2 activity in cancer remain to be established. In the current study, we applied gene knockdown, subcellular fractionation, and stable isotope labeling by amino acids in cell culture-based quantitative proteomic strategies to systematically analyze the KPNA2-regulating protein profiles in an adenocarcinoma cell line. Interaction network analysis revealed that several KPNA2-regulating proteins are involved in the cell cycle, DNA metabolic process, cellular component movements and cell migration. Importantly, E2F1 was identified as a potential novel cargo of KPNA2 in the nuclear proteome. The mRNA levels of potential effectors of E2F1 measured using quantitative PCR indicated that E2F1 is one of the "master molecule" responses to KPNA2 knockdown. Immunofluorescence staining and immunoprecipitation assays disclosed co-localization and association between E2F1 and KPNA2. An in vitro protein binding assay further demonstrated that E2F1 interacts directly with KPNA2. Moreover, knockdown of KPNA2 led to subcellular redistribution of E2F1 in lung cancer cells. Our results collectively demonstrate the utility of quantitative proteomic approaches and provide a fundamental platform to further explore the biological roles of KPNA2 in nonsmall cell lung cancer.