Major lung resection combined with mediastinal lymph node dissection is the standard operation for early-stage non-small cell lung cancer (NSCLC), and lymph node staging is the main predictor of long-term survival. This study aims to identify the prognostic significance of the number of removed lymph nodes in patients with pathologic node-negative (pN0) non-small cell lung cancer (NSCLC). Five hundred thirty-six stage pN0 NSCLC patients, received curative resection between January 2004 and July 2011, were enrolled in this retrospective study. The prognostic factors including the number of removed lymph nodes and clinicopathological characteristics were analyzed. Increasing number of removed lymph nodes was associated with better overall survival (OS) (p = 0.009) of stage pN0 NSCLC patients. The number (p < 0.001) and stations (p = 0.011) of removed lymph nodes, tumor stage (p < 0.001), as well as tumor size (p = 0.017), were independent prognostic factors of stage pN0 NSCLC patients. Overall, patients with more than three stations of examined mediastinal lymph node (p = 0.001) owned better 5-year OS. In subgroup analyses, patients of stage T1 and T2 with more than 11 removed lymph nodes (p = 0.033 and p = 0.011, respectively), and patients of stage T3 with more than 16 removed lymph nodes (p < 0.001) obtained improvements of 5-year OS. The number of removed lymph nodes was not correlated with the incidence of postoperative complications (p = 0.130). The number of removed lymph nodes is an independent prognostic factor for stage pN0 NSCLC patients. Sufficient lymph node dissection is recommended to improve the prognosis of patients with pN0 NSCLC.