Abstract Objectives In mice, low protein (LP) diets increase food intake (FI) thereby energy intake, to compensate for protein deficiency, but mice do not gain fat because total energy expenditure (TEE) is also increased. Fibroblast Growth Factor 21 (FGF21), which is expressed in the liver and in the brain, appears as a key player in these effects. The present study hypothesized that both LP diet but also only lysine or threonine deficiency can modulate FGF21 in the liver and in the hypothalamus that in turn is involved in the control of FI and energy expenditure. Methods Growing rats were fed for 3 weeks: i) LP diets containing 3-5-8-12-15 or 20% of milk protein, or ii) the same diets but supplemented with free amino acids at the level of the 20% protein diet except for lysine or threonine leading to lysine or threonine deficient diets. Body weight and FI were measured daily and energy expenditure were measured by indirect calorimetry. At the end of the experiment, rats were euthanized, tissues and biological fluids were removed and frozen, and body composition as well as gene expression and plasma FGF21 were analyzed. Results Diets with 3 and 5% protein, and diets highly deficient in lysine or threonine (85% and 75%) result in significant growth retardation. LP 3% and 5% induced an increase in relative FI. Surprisingly, an increase in TEE was observed under LP 5% protein, due to an increase in motor activity. Hepatic FGF21 expression is increased at 3 and 5% and strongly decreased at 12, 15 and 20% protein. In contrast, in the hypothalamus, FGF21 expression was significantly lower in LP 3% compared to a 20% protein diet, and for the other LP diets, the values are intermediate. Plasma FGF21 was higher in 3, 5, 8 and 12% protein than in 15 and 20% protein diets. Lysine or threonine deficiency were able to reproduce the effect of LP diet at 3 and 5% whereas at 8%, only the deficiency of threonine was able to reproduce the LP effect. Conclusions These results showed that hepatic and hypothalamic expression FGF21 are inversely affected by protein deficiency. Such situations of deficiency induced an up-regulation of hepatic expression of FGF21 that increased TEE and a down-regulation of hypothalamic expression of FGF21 that could led to hyperphagia. Interestingly, the levels of FGF21 observed for the 3, 5, 8% protein diets could be due at least to lysine and threonine deficiency. Funding Sources ABIES, AlimH department of INRAe.