Sperm-associated antigen 9 (SPAG9) is a recently characterized oncoprotein involved in the progression of several human malignancies. The present study aims to investigate the expression pattern and biological roles of SPAG9 protein in human astrocytoma. SPAG9 expression was analyzed in 105 astrocytoma specimens by immunohistochemistry. We observed negative staining in normal astrocytes and positive staining of SPAG9 in 63 out of 105 (60 %) astrocytoma samples. Overexpression of SPAG9 correlated with tumor grade (p < 0.001). Small interfering RNA knockdown was performed in U251 and U87 cell lines with relatively high SPAG9 expression. Using methylthiazolyldiphenyl-tetrazolium bromide assay and Matrigel invasion assay, we were able to show that SPAG9 depletion in astrocytoma cell lines inhibited cell proliferation and invasion in both cell lines. In addition, mRNA and protein levels of matrix metallopeptidase 9 (MMP9) were downregulated, while the levels of tissue inhibitor of metalloproteinase 1 (TIMP1) and TIMP2 were not changed, indicating that SPAG9 might regulate invasion through MMP9. In conclusion, SPAG9 serves as an important oncoprotein in human astrocytoma by regulating cell proliferation and invasion.