Abstract Background Oct4 is a major transcription factor related to stem cell self-renewal and differentiation. To fulfill its functions, it must be able to enter the nucleus and remain there to affect transcription. KPNA2, a member of the karyopherin family, plays a central role in nucleocytoplasmic transport. The objective of the current study was to examine the association between Oct4 and KPNA2 expression levels with regard to both the clinicopathological characteristics and prognoses of patients with non-small-cell lung cancer (NSCLC). Methods Immunohistochemistry was used to detect the expression profile of Oct4 and KPNA2 in NSCLC tissues and adjacent noncancerous lung tissues. Real-time polymerase chain reaction and western blotting were used to detect the mRNA and protein expression profiles of Oct4 and KPNA2 in lung cancer cell lines. Small interfering RNAs were used to deplete Oct4 and KPNA2 expressions. Double immunofluorescence was used to detect Oct4 expression in KPNA2 knockdown cells. Co-immunoprecipitation was used to detect the interaction of Oct4 and KPNA2. Results Oct4 was overexpressed in 29 of 102 (28.4%) human lung cancer samples and correlated with differentiation (P = 0.002) and TNM stage (P = 0.003). KPNA2 was overexpressed in 56 of 102 (54.9%) human lung cancer samples and correlated with histology (P = 0.001) and differentiation (P = 0.045). Importantly, Oct4 and KPNA2 expression levels correlated significantly (P < 0.01). Expression of Oct4 and KPNA2 was associated with short overall survival. In addition, depleting Oct4 and KPNA2 expression using small interfering RNAs inhibited proliferation in lung cancer cell lines. Real-time polymerase chain reaction and western blotting analysis indicated that reduction of KPNA2 expression significantly reduced mRNA and nucleoprotein levels of Oct4. Double immunofluorescence analysis revealed that nuclear Oct4 signals were reduced significantly in KPNA2 knockdown cells. Co-immunoprecipitation experiments revealed that KPNA2 interacts with Oct4 in lung cancer cell lines. Conclusion Oct4 and KPNA2 play an important role in NSCLC progression. Oct4 nuclear localization may be mediated by its interaction with KPNA2.