Transforming growth factor-β1 (TGFβ-1) signaling is regulated by endocytotic pathway. To clarify the prognostic value of TGFβ-1 and to verify the involvement of endocytosis in drug resistance, we examined the expression of TGFβ-1 and Eps15 homology domain 1 (EHD1) in non-small cell lung cancer (NSCLC) and its association with tumor characteristics and survival of patients with NSCLC. Expression of TGFβ-1 and EHD1 was evaluated by immunohistochemistry in paraffin sections from 105 NSCLC patients. Overall survival (OS) was analyzed by Kaplan-Meier method, log-rank test, and multivariate Cox proportional hazard regression model. Positive immunostaining of TGFβ-1 and EHD1 was detected in 52.38 and 39.05 % of NSCLC samples, respectively. In non-adjuvant chemotherapy-treated group (P = 0.006) and epidermal growth factor receptor (EGFR) (+) group (P = 0.038), patients with TGFβ-1 expression had a longer OS. EHD1 negative expression predicted a longer OS (P = 0.003), especially in EGFR (+) (P = 0.006) and adjuvant chemotherapy-treated patients (P = 0.003). NSCLC patients with concurrent positive TGFβ-1 and negative EHD1 (combined markers) were significantly correlated with better OS (P = 0.001). American Joint Committee on Cancer (AJCC) status and combined markers were independent prognostic indicators for OS (HR (95 % CI) 1.576 (1.112-2.232), P = 0.011 and HR 0.349 (0.180-0.673), P = 0.002, respectively). We identified concordant TGFβ-1 positive and EHD1 negative as a strong favorable prognosis factor in NSCLC. Our results may help us to select and optimize strategies for individualized therapy.