Proline-rich tyrosine kinase 2 (PYK2) is a non-receptor tyrosine kinase, plays different roles in intracellular signaling pathways, that regulates a number of biological processes, such as cell proliferation, differentiation, adhesion and migration, which have been shown to correlate with tumor development and aggression. However, the involvement of PYK2 in human non-small cell lung cancer (NSCLC) has not yet been determined. In the present study, 90 patients with NSCLC (represented by adenocarcinoma and squamous cell carcinoma) were included retrospectively. NSCLC tissues were detected for the expression of PYK2 by immunohistochemistry. Correlation between the expression of PYK2 with the clinicopathological characteristics was analyzed. There were 64% (58 out of 90) of NSCLC patients with higher level of PYK2. Higher expression of PYK2 was significantly correlated with lymph node metastasis (node positive versus node negative, p=0.007). Patients with higher expression of PYK2 had advanced stage of NSCLCs (I+II versus III+IV, p=0.012). Protein level of PYK2 was also examined in 30 of these tumorous samples and matched non-tumorous counterparts by western blotting. PYK2 was apparently up-regulated in NSCLC tissues (tumor versus non-tumor, p=0.000). In the cell studies, extensive expression and activation of PYK2 were both found in higher metastatic BE1 cells. The activity of ERK1/2 in BE1 cells appeared extremely high as well. In conclusion, our results demonstrated that PYK2 is up-regulated in NSCLCs, and the higher expression and activation of PYK2 may play a role in modulating the activity of ERK1/2, and lead to the progression of NSCLC.