Nek6 is a cell cycle regulatory gene, which can control cell proliferation and survival. Recent studies suggested that desregulation of Nek6 expression plays a key role in oncogenesis. This study was aimed to investigate the potential roles of Nek6 in hepatocellular carcinoma (HCC) development. Immunohistochemistry and Western blot analysis was performed for Nek6 in 80 hepatocellular carcinoma samples. The data were correlated with clinicopathological features. The univariate and multivariate survival analyses were performed to determine the prognostic significance of Nek6 in HCC. In addition, Nek6 expression vector was used to detect its role in cell cycle control. Nek6 was overexpressed in hepatocellular carcinoma as compared with the adjacent normal tissue. High expression of Nek6 was associated with histological grade and the level of alpha fetal protein, and Nek6 was positively correlated with proliferation marker Ki-67. Univariate analysis showed that Nek6 expression was associated with poor prognosis. Multivariate analysis indicated that Nek6 and Ki-67 protein expression was an independent prognostic marker for HCC. While in vitro, following release from serum starvation of HuH7 HCC cell, the expression of Nek6 was upregulated. Overexpression Nek6 in Huh7 cell could promote the cell cycle. In conclusion, Nek6 is involved in the pathogenesis of hepatocellular carcinoma. It may be a favorable independent poor prognostic parameter for hepatocellular carcinoma.