Nonalcoholic fatty liver disease (NAFLD) is the most prevalent form of chronic liver disease and is a comorbidity in type 2 diabetes (T2D) mellitus. Mesenchymal stem cell (MSC) is emerging as a potential therapeutic strategy for diabetes and NAFLD through mitochondrial transfer initiated by signaling from injured recipient cells. Thus, in this study, we investigated whether exogenous mitochondrial preconditioning of MSCs could exert superior effects on NAFLD and explore the role of MSCs-mediated mitochondrial transfer into hepatocyte. After free HepG2 mitochondria pretreated, umbilical cord-derived MSCs (UC-MSCs) (mito-MSCs), T2D model mice were infused with equal amounts of MSCs/mito-MSCs via the tail vein once a week for 4 weeks. Body weight and random blood glucose were monitored weekly. After the end of treatment, the mitochondrial transfer level of MSCs before and after pretreatment were monitored by fluorescence tracing. Blood and liver were collected for biochemical and histopathological examinations. The number, morphology, and function of mitochondria in liver tissue were evaluated by tissue electron microscopy and western blot analysis. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to monitor the expression of genes associated with lipid metabolism and regulation pathways. Pretreatment of UC-MSCs enhanced the efficacy of MSCs in lowering blood glucose, liver transaminase, triglyceride levels, and reducing histological damage, which may be related to free mitochondria triggering autophagy of MSCs, which in turn promoted the entry of MSCs mitochondria into the liver tissue of diabetic mice. Exogenous mitochondria could enhance the therapeutic efficacy of MSCs in NAFLD via mediating mitochondrial transfer, which offers a novel strategy for the improving the outcomes of MSCs cell-therapy for diabetes-related NAFLD.