In our previous report, we identified miR-34c-3p as an independent factor contributing to the carcinogenesis of hepatocellular carcinoma (HCC) by targeting NCK Associated Protein 1 (NCKAP1). NCKAP1 has been known to promote the malignancy of cancer cells by disrupting the structural stability of WAS protein family member 1 (WASF1) and is correlated with poor prognosis of patients in several cancer types. Our results, however, show that NCKAP1 is correlated with a favorable outcome in HCC patients. The underlying mechanism of this contradictory phenomenon is unknown. The current study was designed to explore the mechanism of NCKAP1 in HCC. As a result, clinicopathological correlations and results from in vivo and in vitro models indicated that NCKAP1 was a tumor suppressor gene. Cell cycle analysis suggested that NCKAP1 inhibit cells from entering G2/M phase. Western blot analysis showed that WASF1 was barely expressed in HCC cell lines compared to that of breast cancer cell lines, which serve as positive controls. Furthermore, Rb1 and p53 expression was upregulated in cell lines overexpressing NCKAP1. Expression of several cell cycle regulating proteins also varied in the HCC cell lines. In conclusion, although previous studies have identified NCKAP1 as a cell invasion promoter by binding to WASF1, we found that NCKAP1 is a tumor suppress gene that modulates the cell cycle of HCC cell lines by targeting Rb1/p53 regulation.