The study investigates the mechanisms underlying the resistance to diet-induced obesity in germ-free (GF) mice. GF mice, compared to conventionally raised (CONV-R) mice, are protected from obesity after consuming a Western-style, high-fat, sugar-rich diet. This protection is associated with increased levels of phosphorylated AMP-activated protein kinase (AMPK) and its downstream targets involved in fatty acid oxidation in skeletal muscle and liver. The resistance to obesity in GF mice is independent of fasting-induced adipose factor (Fiaf), a circulating lipoprotein lipase inhibitor. However, GF Fiaf−/− mice, lacking Fiaf, exhibit similar levels of phosphorylated AMPK but have reduced expression of genes encoding peroxisomal proliferator-activated receptor coactivator (Pgc-1α) and enzymes involved in fatty acid oxidation. The findings suggest that the gut microbiota influences both sides of the energy balance equation, and that the protection against diet-induced obesity in GF mice involves two complementary but independent mechanisms: elevated levels of Fiaf, which induces Pgc-1α, and increased AMPK activity. These results highlight the importance of considering the gut microbiota in the context of energy balance and obesity.The study investigates the mechanisms underlying the resistance to diet-induced obesity in germ-free (GF) mice. GF mice, compared to conventionally raised (CONV-R) mice, are protected from obesity after consuming a Western-style, high-fat, sugar-rich diet. This protection is associated with increased levels of phosphorylated AMP-activated protein kinase (AMPK) and its downstream targets involved in fatty acid oxidation in skeletal muscle and liver. The resistance to obesity in GF mice is independent of fasting-induced adipose factor (Fiaf), a circulating lipoprotein lipase inhibitor. However, GF Fiaf−/− mice, lacking Fiaf, exhibit similar levels of phosphorylated AMPK but have reduced expression of genes encoding peroxisomal proliferator-activated receptor coactivator (Pgc-1α) and enzymes involved in fatty acid oxidation. The findings suggest that the gut microbiota influences both sides of the energy balance equation, and that the protection against diet-induced obesity in GF mice involves two complementary but independent mechanisms: elevated levels of Fiaf, which induces Pgc-1α, and increased AMPK activity. These results highlight the importance of considering the gut microbiota in the context of energy balance and obesity.