Whether amino acids act on cellular insulin signaling remains unclear, given that increased circulating amino acid levels are associated with the onset of type 2 diabetes (T2D). Here, we report that phenylalanine modifies insulin receptor beta (IRβ) and inactivates insulin signaling and glucose uptake. Mice fed phenylalanine-rich chow or phenylalanine-producing aspartame or overexpressing human phenylalanyl-tRNA synthetase (hFARS) developed insulin resistance and T2D symptoms. Mechanistically, FARS phenylalanylated lysine 1057/1079 of IRβ (F-K1057/1079), inactivating IRβ and preventing insulin from promoting glucose uptake by cells. SIRT1 reversed F-K1057/1079 and counteracted the insulin-inactivating effects of hFARS and phenylalanine. F-K1057/1079 and SIRT1 levels in white blood cells from T2D patients wereare positively and negatively correlated with T2D onset, respectively. Blocking F-K1057/1079 with phenylalaninol sensitizeds insulin signaling and relieveds T2D symptoms in hFARS-transgenic and db/db mice. These findings shed light on the activation of insulin signaling and T2D progression through inhibition of phenylalanylation.