The objectives of this study were to detect the expressions of microRNA-218 (miR-218) in human gastrointestinal stromal tumor (GIST) tissues and cells and explore its effects on the biological features of GIST-T1 cells and the expression of its target gene KIT, so as to provide new insights for GIST treatment. Using quantitative real-time polymerase chain reaction (qRT-PCR), we detected the expressions of miR-218 in the tissues and adjacent tissues of GIST and in the GIST cell lines including GIST882, GIST430, GIST48, and GIST-T1. Forty-eight hours after the miR-218 mimic was transfected into the GIST-T1 cells, the expression of miR-218 in the GIST-T1 cells was detected by qRT-PCR. The effect of miR-218 on the GIST-T1 cell viability was detected using MTT. The effect of miR-218 on the proliferation and apoptosis of GIST-T1 cell was analyzed using flow cytometry. Transwell invasion chamber was applied to detect the effect of miR-218 on the invasion of GIST-T1 cells. KIT was identified to be a target gene of miR-218 by the luciferase reporter enzyme system, and the effect of miR-218 on the expression of KIT protein in cells was determined using Western blotting. As shown by qRT-PCR, compared with that in the GIST adjacent tissue, the expressions of miR-218 in the tumor tissue and GIST cell lines were significantly decreased (P < 0.0001). Compared with the control group, the expression of miR-218 increased significantly in GIST-T1 cells transfected with miR-218 mimic for 48 h (P < 0.01). MTT showed that the cell viability decreased significantly after the overexpression of miR-218 in the GIST-T1 cells (P < 0.01). Flow cytometry showed that the cell proliferation index significantly declined after the overexpression of miR-218 (P < 0.01); meanwhile, the apoptosis of cells also significantly increased (P < 0.01). Detection using the Transwell invasion chamber showed that the number of cells passing through the Transwell chamber significantly dropped after the enhanced expression of miR-218 (P < 0.01). Luciferase reporter gene assay showed that, compared with the control group, the relative luciferase activity significantly declined in the miR-218 mimic transfection group (P < 0.01). Compared with the control group, the expression of KIT protein in the GIST-T1 cells transfected with miR-218 mimic for 48 h significantly decreased (P < 0.01). In conclusion, the expression of miR-218 decreases in human GIST tissue and cell lines. miR-218 can negatively regulate the expression of KIT protein and inhibit the proliferation and invasion of GIST cells. Treatment based on the enhanced expression of miR-218 may be a promising strategy for GIST.