Multiple cancer-associated single-nucleotide polymorphisms (SNPs) have been mapped to conserved sequences within a 500-kilobase region upstream of theMYConcogene on human chromosome 8q24. These SNPs may affect cancer development through altered regulation ofMYCexpression, but this hypothesis has been difficult to confirm. We generated mice deficient in Myc-335, a putativeMYCregulatory element that contains rs6983267, a SNP accounting for more human cancer-related morbidity than any other genetic variant or mutation. In Myc-335 null mice,Myctranscripts were expressed in the intestinal crypts in a pattern similar to that in wild-type mice but at modestly reduced levels. The mutant mice displayed no overt phenotype but were markedly resistant to intestinal tumorigenesis induced by theAPCminmutation. These results establish that a cancer-associated SNP identified in human genome-wide association studies has a functional effect in vivo.