Objective To investigate the protective effect of 1,25(OH) ₂ VD₃ on hepatic mitochondrial injury in ZDF rats and the potential mechanism. Methods Male Zucker rats (5 - 6 weeks old) were randomly divided into three groups according to their weight: control (ZL), model (ZDF), and 1,25(OH) ₂ VD₃ supplementation (ZDF + VD) groups. All rats were treated to 12 weeks of age, and then the related parameters were analyzed. Results ZDF rats treated with 1,25(OH) ₂ VD₃ showed a significant decrease in serum ALT/ AST and liver lipid droplet deposition. Treatment with 1,25(OH) ₂ VD₃ ameliorated mitochondrial injury and increased the expression of mitochondrial biogenesis-related factors SIRT1, PGC-1α, NRF1, and TFAM. Conclusions These findings suggest that 1,25(OH) ₂ VD₃ alleviates diabetic liver injury by improving mitochondrial biogenesis through upregulating SIRT1, PGC-1α, NRF1, and TFAM expression.