Non-alcoholic steatohepatitis (NASH) is a disease in which fatty liver develops independently of alcohol intake and progresses to cirrhosis and liver cancer. Currently, no effective treatment for NASH has been found, and new approaches to elucidate the pathogenesis of the disease are needed. It’s also been suggested that NASH is associated with functional abnormalities in mitochondria, which are involved in lipid metabolism. Our laboratory has successfully established liver organoids from NASH model mice that can reproduce the fibrotic pathology of NASH, and electron microscopic images of NASH liver organoids showed lipid accumulation, mitochondrial deformation, and aggregation compared to normal liver organoids. Therefore, we analyzed the morphological changes and expression levels of fibrosis-related markers in NASH liver organoids upon treatment with Mdivi-1, a mitochondrial mitogen (DRP1) inhibitor. The results showed that Mdivi-1 suppressed the expression of dendritic morphology in NASH liver organoids and decreased the mRNA expression levels of Collagen-I and α-SMA. In addition, when Mdivi-1 was administered long-term to mice fed a NASH-inducing diet, improvement of fatty liver was observed compared to the solvent-fed group. These results suggest that Mdivi-1 may be useful as a therapeutic agent to improve NASH pathology.