Mitochondrial fatty acid oxidation (mFAO) is considered to be essential for driving gluconeogenesis (GNG) during fasting. However, quantitative in vivo data on de novo synthesis of glucose‐6‐phosphate upon acute inhibition of mFAO are lacking. We assessed hepatic glucose metabolism in vivo after acute inhibition of mFAO by 30 mg kg⁻¹ 2‐tetradecylglycidic acid (TDGA) in hypoketotic hypoglycemic male C57BL/6J mice by the infusion of [U‐¹³C]glucose, [2‐¹³C]glycerol, [1‐²H]galactose, and paracetamol for 6 hours, which was followed by mass isotopomer distribution analysis in blood glucose and urinary paracetamol‐glucuronide. During TDGA treatment, endogenous glucose production was unaffected (127 ± 10 versus 118 ± 7 μmol kg⁻¹ minute⁻¹, control versus TDGA, not significant), but the metabolic clearance rate of glucose was significantly enhanced (15.9 ± 0.9 versus 26.3 ± 1.1 mL kg⁻¹ minute⁻¹, control versus TDGA,P < 0.05). In comparison with control mice, de novo synthesis of glucose‐6‐phosphate (G6P) was slightly decreased in TDGA‐treated mice (108 ± 19 versus 85 ± 6 μmol kg⁻¹ minute⁻¹, control versus TDGA, P < 0.05). Recycling of glucose was decreased upon TDGA treatment (26 ± 14 versus 12 ± 4 μmol kg⁻¹ minute⁻¹, control versus TDGA, P < 0.05). Hepatic messenger RNA (mRNA) levels of genes encoding enzymes involved in de novo G6P synthesis were unaltered, whereas glucose‐6‐phosphate hydrolase mRNA expressions were increased in TDGA‐treated mice. Glucokinase and pyruvate kinase mRNA levels were significantly decreased, whereas pyruvate dehydrogenase kinase isozyme 4 expression was increased 30‐fold; this suggested decreased glycolytic activity. Conclusion: Acute pharmacological inhibition of mFAO using TDGA had no effect on endogenous glucose production and only a marginal effect on de novo G6P synthesis. Hence, fully active mFAO is not essential for maintenance of hepatic GNG in vivo in fasted mice.(HEPATOLOGY 2008.)