The Wnt family genes encode multifunctional signaling glycoproteins that are involved in the regulation of a wide variety of normal and pathological processes including tumorigenesis. In order to clarify the clinical significance of the intratumoral Wnt1 expression in non-small cell lung cancer (NSCLC), we performed an immunohistochemical study on the Wnt1 expression in NSCLCs in relation to the tumor proliferation. The intratumoral Wnt1 protein expression appeared in a cytoplasmic staining pattern. Of the 151 NSCLCs studied, 61 carcinomas (40.4%) were Wnt1-positive. Regarding the tumor biology of the intratumoral Wnt1 expression, the Ki-67 proliferation index was significantly higher in Wnt1-positive than in Wnt1-negative tumors (P=0.0062). Furthermore, regarding the expression of c-Myc, one of the proliferation-regulating Wnt targets, the percentage of c-Myc-positive tumor cells was significantly higher in Wnt1-positive than in Wnt1-negative tumors (P=0.0019). The Ki-67 proliferation index was significantly higher in c-Myc-positive than in c-Myc-negative tumors (P=0.0239). The overall survival was significantly lower in patients with Wnt1-positive NSCLCs than in patients with Wnt1-negative NSCLCs (P=0.0003). A Cox regression analysis demonstrated that the Wnt1 status was a significant prognostic factor for NSCLC patients (hazard ratio 1.983; P=0.0061). Our results revealed that the Wnt1 overexpression affects the tumor proliferation in NSCLCs, partly via the upregulation of c-Myc.