Transforming growth factor-β1 (TGF-β1) is known to suppress antitumor immune responses, and its overexpression is closely associated with a poor prognosis in patients with malignant tumors. Moreover, TGF-β1 29T>C genetic polymorphism is known to affect survival among breast cancer patients. The relationship between TGF-β1 polymorphism and the clinicopathological characteristics of non-small cell lung cancer remains unknown, however. The study participants were 91 Japanese patients who underwent curative surgery for adenocarcinoma of the lung. DNA was extracted from tumor samples, and TGF-β1 29T>C genetic polymorphism was investigated using the polymerase chain reaction-restriction fragment length polymorphism method, after which genotype was correlated with clinicopathological factors. There were no differences between the TGF-β1 29TT and 29TC+CC genotypes with respect to age, sex, histological differentiation grade, tumor size, or pathological stage. However, the frequency of nodal metastasis was significantly greater in the TGF-β1 29TC+CC group than the TGF-β1 29TT group. Multivariate logistic regression analysis of lymph node metastasis revealed that male, tumor size, differentiation grade, and TGF-β1 29TC+CC genotypes (hazard ratio, 5.26; 95% CI, 1.03-40.0; P = 0.045) are factors associated with a significantly greater likelihood of developing lymph node metastasis. TGF-β1 29T>C genetic polymorphism is an independent factor associated with lymph node metastasis in adenocarcinoma of the lung.