The pathogenesis of non-alcoholic steatohepatitis (NASH) is poorly understood. Here, relationships between mitochondrial depolarization (mtDepo) and mitochondrial homeostasis were studied in a mouse model of NASH. C57BL/6 mice were fed a Western diet (high fat, fructose and cholesterol) for 2 weeks, 2 months and 6 months, and livers were harvested for histology and biochemical analysis. Hepatic mtDepo was evaluated by intravital multiphoton microscopy. After Western diet feeding, mixed hepatic micro- and macrovesicular steatosis and leukocyte infiltration occurred at 2 weeks and continued to increase afterwards. ALT release, mild necrosis, apoptosis, and ballooning degeneration were present at 2 and 6 months. Smooth muscle α-actin expression increased at 2 weeks and longer, and increased collagen-I expression and mild fibrosis occurred at 6 months. After feeding Western diet for 2 weeks and longer, mtDepo appeared in 50-70% hepatocytes, indicating mitochondrial dysfunction at an early stage of NASH. mtDepo can initiate mitophagy, and mitophagic markers increased at 2 and 6 months. Concurrently autophagic processing became impaired. Oxidative phosphorylation proteins, mitochondrial biogenesis signals, and proteins associated with mitochondrial fission and fusion decreased after 2 months and longer of Western diet. Proinflammatory and profibrotic signaling (NLRP3 inflammasome activation, expression of IL-1, osteopontin and TGF-ß1) also increased in association with mitochondrial stress/dysfunction after Western diet feeding. Taken together, we show that hepatic mtDepo occurs early in mice fed a Western diet, followed by increased mitophagic burden, suppressed mitochondrial biogenesis and dynamics, and mitochondrial depletion. These novel mitochondrial alterations in NASH most likely play an important role in promoting steatosis, inflammation, and progression to fibrosis.