Gastrointestinal stromal tumors (GISTs), are characterized by mutations of the KIT or platelet-derived growth factor receptor-α gene and the constitutive expression of Kit, which is currently being studied as a potential therapeutic target. In this study, we addressed the question of whether the microRNA (miRNA) 221/222 cluster (miR-221/222), which has been shown to be dysregulated in many malignancies, is linked to GIST diagnosis and prognosis, and whether it could provide a basis for possible therapeutic approaches. We analyzed the expression of miR-221 and miR-222 in 54 formalin-fixed and paraffin-embedded GISTs and corresponding peripheral non-tumorous tissue by real-time PCR. The miRNA-expression levels were studied in relation to histomorphological parameters, KIT mutation status and immunohistochemical Kit expression. miR-221 and miR-222, were reduced in most of the GISTs, in contrast to other tumors. No correlation was observed between miR-221/222 expression levels and histomorphological parameters, tumor risk grade, or KIT mutation status. However, we found major differences in miRNA expression among the different groups of immunohistochemical Kit expression, especially between Kit-negative and -positive tumors. The expression levels of miR-221 and miR-222 were significantly repressed in Kit-positive GISTs, compared to normal tissue, whereas Kit-negative GISTs exhibited a completely inverse expression pattern. This study shows for the first time that miR-221 and miR-222 can act as regulators of Kit expression in GISTs and hence reveals a new aspect in the molecular pathogenesis of these tumors. Although miR-221/222 expression does not have an impact on diagnostics, it could be considered as a tool for future therapeutic strategies for GISTs, especially for tumors with secondary resistance to tyrosine kinase inhibitors.