Elevation in homocysteine (Hcy) level is associated with insulin resistance; however, the causality between them and the underlying mechanism remain elusive. Here, we show that Hcy induces insulin resistance and causes diabetic phenotypes by protein cysteine-homocysteinylation (C-Hcy) of the pro-insulin receptor (pro-IR). Mechanistically, Hcy reacts and modifies cysteine-825 of pro-IR in the endoplasmic reticulum (ER) and abrogates the formation of the original disulfide bond. C-Hcy impairs the interaction between pro-IR and the Furin protease in the Golgi apparatus, thereby hindering the cleavage of pro-IR. In mice, an increase in Hcy level decreases the mature IR level in various tissues, thereby inducing insulin resistance and the type 2 diabetes phenotype. Furthermore, inhibition of C-Hcy in vivo and in vitro by overexpressing protein disulfide isomerase rescues the Hcy-induced phenotypes. In conclusion, C-Hcy in the ER can serve as a potential pharmacological target for developing drugs to prevent insulin resistance and increase insulin sensitivity.