Background: Mediastinal lymph node staging (N-staging) is essential to optimize the treatment in non-small cell lung cancer (NSCLC). Transesophageal endoscopic ultrasonography-guided fine needle aspiration (EUS-FNA) has recently been introduced as a complementary method. However, in most reports, EUS-FNA has been performed in patients who have demonstrated enlarged lymph nodes (LNs) on CT findings. The yield of EUS/EUS-FNA in patients without enlarged mediastinal LNs by CT has so far only been evaluated in a few reports. Aims: Our aim was to compare the diagnostic accuracy of CT and EUS with or without EUS-FNA (EUS/EUS-FNA) prospectively, for N-stage in all patients with potentially resectable NSCLC, including patients with and without mediastinal LN enlargement based on CT findings. Methods: Eighty consecutive patients with potentially resectable NSCLC based on CT findings were enrolled in this prospective comparative study, and underwent EUS/EUS-FNA. Results: Pathological N-stage was established in 78 patients, while in another 2 cases, malignant pleural effusion was proven by EUS-FNA, and we avoided further N-staging. In the 78 patients, the prevalence of malignant mediastinal LNs was 21%. The accuracy of EUS/EUS-FNA (91%) was significantly higher than that of CT (71%). The negative predictive value of EUS/EUS-FNA was 90%. In addition, EUS-FNA identified 2 patients as N3 disease in 56 patients without mediastinal LN involvement on CT. Conclusions: EUS/EUS-FNA gave more accurate N-staging in patients with possibly resectable NSCLC than CT, and is thus considered to be useful to determine the optimal treatment strategy.