Background and Purpose: Excessive mitochondrial fission was observed in diabetic kidney disease (DKD). Phosphoglycerate mutase family member 5 (PGAM5) plays an important role in mitochondrial fission by dephosphorylating the DRP1S637. Whether PGAM5 participates in the mitochondrial fission in diabetic renal tubular injury is unknown. Clinical trials have observed encouraging effect of Sodium-glucose cotransporter 2 (SGLT2) inhibitors on DKD though the underling mechanisms remain unclear. Experimental Approach: We use KK-Ay mice as diabetic model and Empa was administrated by oral gavage. The mitochondrial fission and expressions of p-AMPK, SP1, PGAM5 and DRP1-S637 were tested. We also examined these changes in HK2 cells that cultured in normal glucose (NG), high glucose (HG) and high glucose+Empa (HG+Empa) environment. Then we verified our deduction using AMPK activator, inhibitor, si-SP1 and si-PGAM5. Lastly, we testified the interaction between SP1 and the PGAM5promotor by CHIP assay. Key Results: The mitochondrial fission and the expression of SP1, PGAM5 increased and the expression of p-AMPK, DRP1-S637 decreased in diabetic or HG environment. These changes were all reversed in Empa or AICAR treated groups. These reversal effect of Empa could be diminished by Compound C. Either si-SP1 or si-PGAM5 could alleviated the mitochondrial fission without affection on AMPK phosphorylation. Finally, the CHIP assay confirmed the interaction between SP1 and the PGAM5 promotor. Conclusions and Implications: The PGAM5 aggravates the development of diabetic renal tubular injury and the Empa can improve the DKD by alleviating mitochondrial fission via AMPK/SP1/PGAM5 pathway.