HMGB3 overexpression has been reported in a variety of human cancers. However, the role of HMGB3 in human non-small cell lung cancer (NSCLC) remains unclear. In this study, the HMGB3 expression was examined at mRNA and protein levels by quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR), Western blotting, and immunohistochemistry in NSCLC tissues and adjacent non-cancerous tissues. Statistical analyses were applied to test the associations between HMGB3 expression, clinicopathologic factors, and prognosis. Western blotting and qRT-PCR showed that the expression levels of HMGB3 mRNA and protein were both significantly higher in NSCLC tissues than those in non-cancerous tissues. Immunohistochemistry analysis showed that HMGB3 expression was significantly correlated with tumor grade, tumor size, clinical stage, and lymph node metastases. The results of Kaplan-Meier analysis indicated that a high expression level of HMGB3 resulted in a significantly poor prognosis of NSCLC patients. Importantly, multivariate analysis showed that high HMGB3 expression was an independent prognostic factor for NSCLC patients. In sum, our data suggest that HMGB3 plays an important role in NSCLC progression, and that overexpression of HMGB3 in tumor tissues could be used as a potential prognostic marker for patients with NSCLC.