Folate is required for biological methylation and nucleotide synthesis, aberrations of which are thought to be the mechanisms that enhance colorectal carcinogenesis produced by folate inadequacy. These functions of folate also depend on the availability of other B-vitamins that participate in “onecarbon metabolism,” including B2, B6 and B12. Our study therefore investigated whether combined dietary restriction of these vitamins amplifies aberrations in the epigenetic and genetic integrity of the p53 gene that is induced by folate depletion alone. Ninety-six mice were group pair-fed diets with different combinations of B-vitamin depletion over 10 weeks. DNA and RNA were extracted from epithelial cells isolated from the colon. Within the hypermutable region of p53 (exons 5–8), DNA strand breaks were induced within exons 6 and 8 by folate combined with B2, B6 and B12 restriction (p < 0.05); such effects were not significantly induced by mild folate depletion alone. Similarly, a minor degree of hypomethylation of exon 6 produced by isolated folate depletion was significantly amplified (p ≤ 0.05) by simultaneous depletion of all 4 B-vitamins. Furthermore, the expression of p53 and MDM2 were significantly decreased (p ≤ 0.05) by the combined depletion state but not by folate depletion alone. These data indicate that inadequacies of other 1-carbon vitamins may amplify aberrations of the p53 gene induced by folate depletion alone, implying that concurrent inadequacies in several of these vitamins may have added tumorigenic potential beyond that observed with isolated folate depletion.