〔Abstract〕 Objective To rescueSRSF9 gene in human glioma U87SRSF9 knockout monoclonal cell line using CRISPR/Cas9 system and to research its function. Methods SRSF9-WT andSRSF9-sgMu overexpression vector which was synonymous mutated 5 bases on site ofSRSF9-WT were conducted and packaged into lentivirus. U87SRSF9 knockout monoclonal cell lines were infected with lentivirus to constructSRSF9 gene rescue cell lines. Western blot and immunofluorescence assays were used to detect the localization of rescued protein, proliferation experiment and tumor stem cell sphere formation assay were used to detect the rescue functions of rescued gene. Results SRSF9-WT andSRSF9-sgMu overexpression vector were successfully constructed and packaged into lentivirus. Western blot and immunofluorescence assays confirmed that the rescue was successful and therescuedSRSF9 protein was predominantly expressed in nucleus. Theresultsofproliferation and tumor stem cell sphere formation assays showed that the rescuedSRSF9 could also rescue its function(P<0.05). Conclusion SRSF9-sgMu can successfully rescue the expression ofSRSR9 and its function of proliferation and tumor stem cell sphere formation in human glioma U87SRSF9 knockout monoclonal cell line.