〔Abstract〕 Objective To investigated the effects of gene-interfering long-chain non-coding RNA(lncRNA) SPRY4-IT1 on stem cell-like characteristics and mitochondrial membrane potential changes in gallbladder cancer cell line GBC-SD. Methods shRNA-SPRY4-IT1 shRNA was designed and synthesized. shRNA-SPRY4-IT1 was transfected into GBC-SD cells,and the expression level of SPRY4-IT1 was detected by RT-PCR. BrdU staining was used to detect cell proliferation. Cell apoptosis was detected by flow cytometry. Tumor cell pelleting assay was used to detect the characteristics of tumor stem cells. The changes of mitochondrial membrane potential were detected by flow cytometry. Western blot was used to detect the protein expression of dry tumor characteristic markers and mitochondrial damage markers. Results sh RNA-SPRY4-IT1 was transfected into GBC-SD cells,the expression level of SPRY4-IT1 decreased. Interference with SPRY4-IT1 inhibited proliferation of GBC-SD cells,improved mitochondrial membrane potential and promoted apoptosis of GBC-SD cells,reduced stem cell-like properties of gallbladder cancer,inhibited the expression of SOX2,OCT4,CD44,c-Myc,Bcl-2 and promoted the expression of cleaved Caspase-3,cleaved Caspase-9,Bax. Conclusion Gene interference with lncRNA SPRY4-IT1 can reduce the characteristics of GBC-SD cancer stem cells in gallbladder cancer cells,increase mitochondrial membrane potential,and promote apoptosis of gallbladder cancer cell line GBC-SD.