Colorectal cancer (CRC) onset is profoundly affected by Western diet. Here, we report that high-fat (HF) diet-induced, organ-specific colonic lysine homocysteinylation (K-Hcy) increase might promote CRC onset by impeding DNA damage repair. HF chow induced elevated methionyl-tRNA synthetase (MARS) expression and K-Hcy levels and DNA damage accumulation in the mouse and rat colon, resulting in a phenotype identical to that of CRC tissues. Moreover, the increased copy number of MARS, whose protein product promotes K-Hcy, correlated with increased CRC risk in humans. Mechanistically, MARS preferentially bound to and modified ataxia-telangiectasia and Rad3-related protein (ATR), inhibited ATR and its downstream effectors checkpoint kinase-1 and p53, and relieved cell-cycle arrest and decreased DNA damage-induced apoptosis by disrupting the binding of ATR-interacting protein to ATR. Inhibiting K-Hcy by targeting MARS reversed these effects and suppressed oncogenic CRC cell growth. Our study reveals a mechanism of Western-diet-associated CRC and highlights an intervention approach for reversing diet-induced oncogenic effects. Display omitted •High-fat diet induces colonic lysine homocysteinylation (K-Hcy) catalyzed by MARS•Increased MARS copy number is a risk factor for human colorectal cancer (CRC)•K-Hcy impedes DNA damage repair in CRC•K-Hcy inhibition decreases high-fat-induced oncogenic effects Wang et al. find that high-fat diet induces K-Hcy through elevating colonic MARS expression. They show that K-Hcy modification of ATR suppresses DNA damage repair to result in an accumulation of colonic DNA damage.