Objectives: Sodium–glucose co-transporter 2 (SGLT2) inhibitors improve renal and cardiovascular outcomes in patients with chronic kidney disease (CKD) regardless of type 2 diabetes mellitus(T2DM). However, the mechanisms underlying these beneficial effects remains to be elucidated. Mitochondrial dysfunction plays a pivotal role in the pathophysiology of T2DM and CKD. In this work, we aimed to investigate SGLT2 inhibitor improves mitochondrial dysfunction in patients with T2DM or CKD. Methods: We prospectively recruited SGLT2 inhibitor naïve patients with T2DM (n = 16) or non-diabetic CKD (n = 16). Copy numbers of urinary mitochondrial DNA (mtDNA) nicotinamide adenine dinucleotide dehydrogenase subunit-1 (mtND-1) and cytochrome-c oxidase 3 (mtCOX-3) were measured using quantitative polymerase chain reaction at baseline and after 3 months of treatment with SGLT2 inhibitors (empagliflozin in T2DM and dapagliflozin in non-diabetic CKD). Results: The mean age was 52.16 ± 14.44 and 37.5% were female. Immunoglobulin A nephropathy was the most common underlying kidney disease (50%) in the non-diabetic CKD group. Estimated glomerular filtration rate was lower (P <0.001) and urine albumin-to-creatinine ratio was higher (P = 0.001) in the CKD group compared to the T2DM group, whereas the baseline urinary mtDNA copy numbers were comparable. Urinary copy numbers of mtND-1 and mtCOX-3 decreased after SGLT2 inhibitor treatment in both T2DM and non-diabetic CKD group (P <0.001 for mtND-1 and mtCOX-3, respectively). The amount of reduction of urinary mtDNA copy number did not differ according to the class of SGLT2 inhibitor (P = 0.985 for mtND-1, P = 1.000 for mtCOX-3). Conclusions: SGLT2 inhibitor reduces the elevated urinary mtND-1 and mtCOX-3 copy numbers in patients with T2DM or CKD. Our results suggest that SGLT2 inhibitor improves mitochondrial injury regardless of diabetes.