In this study, we examined the expression of osteopontin (OPN), αvβ3 and Pim-1 in non-small cell lung cancer (NSCLC) and investigated the potential clinical implications of their expression patterns in NSCLC. Immunohistochemical assays were used to examine the protein expression of OPN, αvβ3 and Pim-1 in 208 NSCLC samples and their adjacent normal lung tissue specimens. Statistical analyses were performed to evaluate the relationships between OPN, αvβ3 and Pim-1 expression patterns, and their association with the clinical-pathological parameters of NSCLC patients. In NSCLC tissues, the positive rates of OPN, αvβ3 and Pim-1 expression were 67.8% (141/208), 76.0% (158/208) and 58.7% (122/208), respectively. However, in the adjacent normal lung tissues, the positive rates of OPN, αvβ3 and Pim-1 were 20.2% (42/208), 24.0% (50/208) and 14.9% (31/208), respectively. The differences in the positive expression rates of OPN, αvβ3 and Pim-1 between NSCLCs and the adjacent normal lung tissues were all significant (P<0.01). Additionally, the positive expression of OPN, αvβ3 and Pim-1 in NSCLCs was associated with an increase in pathological grade, lymph node metastasis and advanced clinical stage (all P<0.01). Furthermore, associations between the expression of OPN and αvβ3, OPN and Pim-1, and αvβ3 and Pim-1 were also observed in our NSCLC cohort (all P<0.01). The OPN, αvβ3 and Pim-1 proteins are frequently overexpressed in NSCLC and are associated with some clinicopathologic variables that are of known prognostic importance in NSCLC, suggesting that they may play an important role in the development and/or progression of NSCLC.