The homeobox gene NKX6.1 was recently identified in cervical tumors. This study was designed to explore the clinical and prognostic significance of NKX6.1 further in patients with primary hepatocellular carcinoma (HCC). The expression levels of NKX6.1 were examined using real-time PCR, Western blotting, and immunohistochemistry in HCC cell lines and HCC tissues. The invasion capability of cell lines following silencing or overexpression of NKX6.1 was investigated by Transwell assay. Cells proliferation was tested by MTT assays. Epithelial-mesenchymal transition (EMT) marker expression levels were detected in relation to NKX6.1 expression. Correlation between NKX6.1 immunohistochemical staining, clinicopathologic parameters, and follow-up data of HCC patients was analyzed statistically. NKX6.1 expression was higher in HCC tissues compared to the adjacent noncancerous tissue. NKX6.1 overexpression was significantly correlated with tumor size, tumor differentiation, clinical stage, metastasis, and relapse. Kaplan-Meier analysis revealed that NKX6.1 overexpression was related to unfavorable 5-year disease-free survival and overall survival. Importantly, multivariate analysis indicated that NKX6.1 overexpression was an independent unfavorable marker for overall survival. Moreover, a significant relationship was observed between NKX6.1 and EMT marker expression levels, and NKX6.1 knockdown inhibited cell invasion, and overexpression of NKX6.1 promotes cell proliferation in vitro. NKX6.1 is upregulated in HCC and is a reliable prognostic marker for patients with HCC.