Dynamin-related protein 1 (Drp1)-mediated mitochondrial dysfunction is associated with synaptic injury in the diabetic brain. However, the dysfunctional mitochondria by Drp1 deletion in the diabetic brain are poorly understood. Here, we investigated the effects of neuron-specific Drp1 deletion on synaptic damage and mitophagy in the hippocampus of a high-fat diet (HFD)/streptozotocin (STZ)-induced diabetic mice. HFD/STZ-induced diabetic mice exhibited metabolic disturbances and synaptic damages. Floxed Drp1 mice were crossed with Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα)-Cre mice, to generate neuron-specific Drp1 knockout (Drp1cKO) mice, which showed marked mitochondrial swelling and dendritic spine loss in hippocampal neurons. In particular, diabetic Drp1cKO mice exhibited an increase in dendritic spine loss and higher levels of oxidative stress and neuroinflammation compared with diabetic wild-type (WT) mice. Diabetic WT mice generally displayed increased Drp1-induced small mitochondrial morphology in hippocampal neurons, but large mitochondria were prominently observed in diabetic Drp1cKO mice. The levels of microtubule-associated protein 1 light-chain 3 and lysosomal-associated membrane protein 1 proteins were significantly increased in the hippocampus of diabetic Drp1cKO mice compared with diabetic WT mice. The inhibition of Drp1 adversely promotes synaptic injury and neurodegeneration in the diabetic brain. The findings suggest that the exploratory mechanisms behind Drp1-mediated mitochondrial dysfunction could provide a possible therapeutic target for diabetic brain complications.