Insulin resistance (IR) plays a major role in the pathogenesis of abdominal obesity, hypertension, coronary heart disease, atherosclerosis and diabetes. miR21 and TGF-beta/smads is closely related to IR. However, it remained elusive whether metformin improved skeletal muscle insulin resistance (IRSM) by regulating miR-21 and its target signal TGF-beta 1/smads expression. In this study, high-fat diet rats with IR model and IR-skeletal muscle L6 cells (L6-SMCs) model were established, insulin sensitive index (ISI) and Homeostasis model assessment of IR (HOMA-IR) were applied, miR-21 and TGF-beta 1/smads mRNA expression were examined by RT-PCR, smad3 and smad7 protein were detected by western-blotting and laser scanning confocal microscopy (LSCM), the valid target of miR-21 was detected by luciferase reporter gene assay. Here, we found that metformin dose-dependently decreased miR21 expression, accompanied by the decrease of HOMA-IR and the increase of HOMA-ISI. Luciferase report gene assay showed that smad7 was an effective target of miR21. miR-21 overexpression directly downregulated smad7 and indirectly upregulated smad3 expression. Interestingly, miR-21 expression positively correlated with HOMA-IR and negatively correlated with HOMA-ISI. In conclusion, our results demonstrated that metformin improved IRSM by inhibiting miR-21 expression, and that miR-21 may be one of the therapeutic targets for IR.