Sperm-associated antigen 9 (SPAG9) was reported as a novel biomarker for several cancers and associated with the malignant behavior of cancer cells. However, its expression pattern and biological role in human hepatocellular carcinoma (HCC) have not been reported. In the present study, we analyzed SPAG9 expression in human HCC tissues by immunohistochemistry and found that SPAG9 overexpression is correlated with tumor stage (p < 0.001), tumor multiplicity (p = 0.019), tumor size (p = 0.034), AFP levels (p = 0.006), and tumor relapse (p = 0.0017). Furthermore, SPAG9 overexpression is correlated with poor overall survival (p < 0.001) and relapse-free survival (p = 0.002). Transfection of SPAG9 small interfering RNA (siRNA) was performed in Bel-7402 cell line. Colony formation and MTT showed that SPAG9 siRNA knockdown inhibited HCC cell proliferation. We also found that SPAG9 depletion could increase cell apoptosis. In addition, the level of cyclin D1 and cyclin E protein expression was downregulated after siRNA treatment. In conclusion, SPAG9 is overexpressed in human HCC and serves as a prognostic marker. SPAG9 contributes to cancer cell growth through regulation of cyclin proteins.