Folate is an essential water soluble B-vitamin required for DNA synthesis and methylation. Folate deficiency has been associated with some cancers, male subfertility, and causes megaloblastic anemia and neural tube defects. Using a transgenic mouse model (MutaMouse), we investigated the effects of folic acid (FA) deficient, control and supplemented diets on somatic and germline DNA mutation frequency and genome instability in male mice. Inadequate FA intake induced micronuclei formation in RBCs and DNA mutations in bone marrow. In contrast to previous studies, deficient FA intake had no effect on sperm counts or mutations in sperm DNA. Supplemental FA intake had no effect on somatic or germ cell mutations or the induction of mutations by ENU. This study highlights the importance of having an adequate FA intake to ensure DNA integrity and prevent chromosomal damage and also alleviates hypothetical concerns related to high intakes of FA in the Canadian population.