Metformin is a well‐known AMP‐activated protein kinase (AMPK) activator, and it has been shown to inhibit organ fibrosis. Whether AMPKα2 mediates metformin protection against renal fibrosis remains unknown. Here, we aimed to investigate the role of the AMPKα2 isoform in mediating the inhibitory effect of metformin on renal fibrosis. Unilateral ureteral obstruction (UUO) was used to induce renal fibrosis in wild‐type (WT) and AMPKα2 knockout (AMPKα2−/−) mice. Metformin treatment was initiated 3 days before UUO and was continued until 7 days after UUO. In WT mice, metformin significantly inhibited UUO‐induced renal fibrosis. In AMPKα2−/− mice, metformin also tended to inhibit UUO‐induced renal fibrosis. Specifically, metformin significantly reduced UUO‐induced transforming growth factor β1 (TGFβ1) mRNA and protein expression in WT mice but not in AMPKα2−/− mice. In contrast, metformin reduced UUO‐induced TGFβ1 downstream Smad3 phosphorylation in both WT and AMPKα2−/− mice, suggesting that this regulation occurs in an AMPKα2‐independent manner. In conclusion, the underlying mechanisms for the protective effects of metformin against renal fibrosis include AMPKα2‐dependent targeting of TGFβ1 production and AMPKα2‐independent targeting of TGFβ1 downstream signalling. In this regard, metformin has an advantage over other AMPK activators for the treatment of renal fibrosis.