During fasting, mitochondrial fatty-acid β-oxidation (mFAO) is essential for the generation of glucose by the liver. Children with a loss-of-function deficiency in the mFAO enzyme medium-chain acyl-Coenzyme A dehydrogenase (MCAD) are at serious risk of life-threatening low blood glucose levels during fasting in combination with intercurrent disease. However, a subset of these children remains asymptomatic throughout life. In MCAD-deficient (MCAD-KO) mice, glucose levels are similar to those of wild-type (WT) mice, even during fasting. We investigated if metabolic adaptations in the liver may underlie the robustness of this KO mouse. WT and KO mice were given a high- or low-fat diet and subsequently fasted. We analyzed histology, mitochondrial function, targeted mitochondrial proteomics, and transcriptome in liver tissue. Loss of MCAD led to a decreased capacity to oxidize octanoyl-CoA. This was not compensated for by altered protein levels of the short- and long-chain isoenzymes SCAD and LCAD. In the transcriptome, we identified subtle adaptations in the expression of genes encoding enzymes catalyzing CoA- and NAD(P)(H)-involving reactions and of genes involved in detoxification mechanisms. We discuss how these processes may contribute to robustness in MCAD-KO mice and potentially also in asymptomatic human subjects with a complete loss of MCAD activity.