Objective:To explore whether miR-335 has its own transcription unit and effects of rosiglitazone and insulin on transcriptional activity of MiR-335 promoter regions. Methods: We cloned different fragments from upstream sequences of miR-335 in MEST intron by PCR. Then, the luciferase activity of these different plasmids was detected by dual-luciferase reporter system. After the stimulation of 1 μmol/L rosiglitazone and insulin of different concentration, the transcriptional activity change of fragments from upstream sequences of miR-335 was examined. Results: Different miR-335 promoter regions had different transcription activity, and the region of about 600 basic groups in miR-335 upstream exhibited the highest activity. The activity of miR-335 promoter in untreated HEK293T cells showed no statistical significance compared to the baseline value. The levels of miR-335 promoter activity showed significant elevation after the intervention of 1 μmol/L rosiglitazone (P < 0.01). The activity of miR-335 promoter was a little elevated with the stimulation of 10-12 mol/L insulin (P < 0.05). Meanwhile, the levels of miR-335 promoter activity had no statistical significance when the concentration of insulin increased. Conclusion: About 600 basic groups in miR-335 upstream were major promoter regions. Meanwhile, miR-335 may play an important role in the development of obesity and insulin resistance via its own transcription mechanism.