Mitochondrial cholesterol accumulation is a hallmark of alcoholic and non-alcoholic fatty liver diseases and impairs the function of specific solute carriers through changes in membrane physical properties. However, its impact on mitochondrial respiration and organization of respiratory supercomplexes has not been determined so far. Here we fed mice a cholesterol-enriched diet (HC) supplemented with sodium cholate to examine the effect of cholesterol in mitochondrial function. HC feeding increased liver cholesterol content, which downregulated Srebp2 and Hmgcr expression, while sodium cholate administration decreased Cyp7a1 and Cyp8b1 mRNA levels, suggesting the downregulation of bile acid synthesis through the classical pathway. HC-fed mice exhibited increased expression of Stard1 and Mln64 and enhanced mitochondrial free cholesterol levels (2–3 fold), leading to decreased membrane fluidity. Mitochondria from HC-fed mice displayed increased cholesterol loading in both outer and inner mitochondrial membranes. Cholesterol loading decreased complex I and complex II-driven state 3 respiration and mitochondrial membrane potential. Decreased respiratory and uncoupling control ratio from complex I was also observed after in situ enrichment of mouse liver mitochondria with cholesterol or enantiomer cholesterol, the mirror image of natural cholesterol. Moreover, in vivo cholesterol loading decreased the level of complex III₂ and the assembly of respiratory supercomplexes I₁+III₂+IV and I₁+III₂. Moreover, HC feeding caused oxidative stress and mitochondrial GSH (mGSH) depletion, which translated in hepatic steatosis and liver injury, effects that were rescued by replenishing mGSH with GSH ethyl ester. Overall, mitochondrial cholesterol accumulation disrupts mitochondrial functional performance and the organization of respiratory supercomplexes assembly, which can contribute to oxidative stress and liver injury.