ObjectiveTo study the expression and role of homeobox transcription antisense intergenic ribose nucleic acid (HOTAIR) in CD133-positive gastric cancer cells, which was classified to long non-coding RNA (LncRNA). MethodsImmune magnetic cell sorting (MACS) was performed to sort CD133-positive and CD133-negative cells of KATO-Ⅲgastric cancer cells, then reverse transcription polymerase chain reaction (RT-PCR) was performed to detect the expressions of HOTAIR mRNA and CD133 mRNA. After the intervention of small interfering RNA (siRNA) for CD133-positive KATO-Ⅲcells, RT-PCR method was performed to detect the expression of HOTAIR mRNA to select siRNA who had the best silent effect. The selected-siHOTAIR was used to silent the expression of HOTAIR, then the expressions of CD133 mRNA, E-cadherin mRNA, and N-cadherin mRNA were detected by RT-PCR. At last, Transwell experiments were performed to detect the migration ability and invasion ability. Results①?RT-PCR test results showed that, the expression levels of CD133 mRNA and HOTAIR mRNA in CD133-positive group were significantly higher than those of CD133-negative group and no separation group (P < 0.05).②?After interference of siHOTAIR, the expression levels of HOTAIR mRNA in siHOTAIR1 group, siHOTAIR2 group, and siHOTAIR3 group were all significantly lower than those of blank control group and negative control group (P < 0.05), and the expression levels of HOTAIR mRNA in siHOTAIR2 group was lower than those of siHOTAIR1 group and siHOTAIR3 group (P < 0.05). The results indicated that siHOTAIR2 had the best interference efficiency.③?The expression levels of CD133 mRNA and N-cadherin mRNA in siHOTAIR2 group were lower than those corresponding indicators of blank control group and negative control group (P < 0.05), but the expression level of E-cadherin mRNA was higher than those of blank control group and negative control group (P < 0.05). Transwell experiment results showed that, number of cells which through the cell membrane in siHOTAIR2 group was lower than those of blank control group and negative control group (P < 0.05). ConclusionThe expression of HOTAIR mRNA in CD133-positive KATO-Ⅲgastric cancer cells was higher than that of CD133-negative cells, interfering the expression of HOTAIR mRNA can reduce the expression of CD133 mRNA in CD133-positive KATO-Ⅲgastric cancer cells, and can inhibit cell migration and invasion.