〔Abstract〕 Objective To establish SNHG9-knocked out U251 cells by combination of CRISPR/Cas9 and single cell sorting through flow cytometry, and test the effect of SNHG9 deletion on cell proliferation. And to explore the potential mechanism that SNHG9 involved by bioinformatic analysis. Methods Two targets were separately designed for Cas9 at the genomic sites upstream and downstream of SNHG9 gene, the two pairs of primers that guide the synthesis of sgRNA were annealed and cloned into px330-EGFP, and U251 cell were co-transfected with the two successfully constructed plasmids followed by single cell sorting into 96-well plates. Cell clones were raised and the genotypes were identified Gby PCR and sequencing, SNHG9-deleted clone cells were selected for subsequent experiments. Effect of SNHG9 deletion on cell proliferation was assessed by RTCA. Expression of lncRNA SNHG9, its co-expressed genes and their enrichment analysis were performed by online tools. Results Effective targets were designed and plasmid of SNHG9 knockout was successfully constructed. 8 clones survived after single cell sorting and culture, from which 1 clone with SNHG9 totally deleted was obtained. The proliferation capacity of U251 cells significantly decreased after SNHG9 knocking-out(P<0.05). SNHG9 was overexpressed glioblastoma multiforme(GBM) samples when compared with normal tissues, the co-expressed genes most significantly enriched in mitochondrion related functions. Conclusion The SNHG9-deleted U251 clone was successfully established; SNHG9 knocking-out suppressed the proliferation of U251 cells; bioinformatic analysis showed that SNHG9 might involve in the regulation of mitochondrial function.