Germ-free (GF) mice are protected against diet-induced obesity, unlike mice with a gut microbiota. This protection is linked to increased phosphorylated AMP-activated protein kinase (AMPK) and fatty acid oxidation in skeletal muscle and liver. GF mice also have elevated levels of fasting-induced adipose factor (Fiaf), which induces peroxisomal proliferator-activated receptor coactivator 1α (Pgc-1α), enhancing fatty acid metabolism. However, GF mice lacking Fiaf are not protected from diet-induced obesity, indicating that Fiaf plays a key role in this resistance. The gut microbiota influences energy balance by modulating both energy intake and expenditure. It suppresses Fiaf expression in the gut epithelium, which normally inhibits lipoprotein lipase (LPL), thereby increasing fat storage. In contrast, GF mice have reduced LPL activity, leading to decreased fat storage. The microbiota also affects AMPK activity, which promotes fatty acid oxidation. These findings suggest that the gut microbiota can influence both sides of the energy balance equation, highlighting the importance of considering the gut microbiota in the context of the human metabolome. The study underscores the role of the gut microbiota in regulating energy homeostasis and provides insights into potential therapeutic targets for obesity prevention and treatment.Germ-free (GF) mice are protected against diet-induced obesity, unlike mice with a gut microbiota. This protection is linked to increased phosphorylated AMP-activated protein kinase (AMPK) and fatty acid oxidation in skeletal muscle and liver. GF mice also have elevated levels of fasting-induced adipose factor (Fiaf), which induces peroxisomal proliferator-activated receptor coactivator 1α (Pgc-1α), enhancing fatty acid metabolism. However, GF mice lacking Fiaf are not protected from diet-induced obesity, indicating that Fiaf plays a key role in this resistance. The gut microbiota influences energy balance by modulating both energy intake and expenditure. It suppresses Fiaf expression in the gut epithelium, which normally inhibits lipoprotein lipase (LPL), thereby increasing fat storage. In contrast, GF mice have reduced LPL activity, leading to decreased fat storage. The microbiota also affects AMPK activity, which promotes fatty acid oxidation. These findings suggest that the gut microbiota can influence both sides of the energy balance equation, highlighting the importance of considering the gut microbiota in the context of the human metabolome. The study underscores the role of the gut microbiota in regulating energy homeostasis and provides insights into potential therapeutic targets for obesity prevention and treatment.