Dietary protein dilution (DPD) promotes metabolic health but the precise nutritional components driving this response remain ill defined. Here we definitely demonstrate that, independent of dietary carbohydrate supply, dietary amino acids (AA) are sufficient and necessary to drive the increases in metabolic inefficiency, systemic insulin sensitivity, and serum/liver FGF21 to DPD. In particular, the restriction of dietary essential AA (EAA) supply, but not non-essential AA, drives the systemic metabolic response to total AA deprivation. Furthermore, of the nine EAA, systemic deprivation of the strictly EAA, THR and TRP, are both adequate and necessary to confer the systemic metabolic response to both diet-, and genetic AA-transport loss-, driven AA restriction. Of note, serum THR is also lower in response to a naturally low-protein diet fed to humans, and THR was identified as the most limiting EAA in this diet according to exome matching analysis. Taken together, our studies demonstrate that the restriction of the strictly EAA, particularly THR and TRP, are sufficient and necessary to confer the systemic metabolic effects of DPD.