Background. Malignant melanoma (MM) is an aggressive malignancy, which accounts for 80% of skin cancer-related deaths and is notably resistant to conventional chemotherapeutic agents. One of the most common treatments for melanoma is surgery, followed by various combinations of chemotherapy drugs. Aim. To investigate the role of microRNA (miR)-203 in sensitivity of MM cells to the chemotherapy drug temozolomide (TMZ). Methods. Using quantitative reverse transcription PCR, we measured the expression of miR-203 in an MM cell line. Cell viability of MM cells in response to TMZ treatment was measured by MTT assay. Glutamine metabolism and level of glutaminase (GLS) were assessed. Results. We found that miR-203 was significantly downregulated by TMZ treatment in human MM cells. In addition, miR-203 expression was lower in TMZ-resistant MM cells compared with parental cells. Interestingly, glutamine metabolism and GLS expression were higher in TMZ-resistant cells, and TMZ-resistant cells exhibited more glutamine dependency than TMZ-sensitive MM cells. We also identified GLS as a downstream target gene of miR-203, which binds directly to the 3 untranslated region of GLS. Overexpression of miR-203 was associated with decreased GLS expression and sensitization to TMZ in vitro. Re-expression of GLS in miR-203 overexpressing MM cells markedly rescued miR-203-mediated suppression of these events. Finally, we found a significant negative correlation between miR-203 and GL, with downregulation of miR-203 and upregulation of GLS in tissues from patients with MM. Conclusion. Taken together, our results demonstrate that overexpression of miR-203 sensitizes MM cells to TMZ by targeting GLS, providing new insights into the development of anti-tumour agents for patients with chemotherapy-resistant MM.