Epidemiological studies suggest an inverse association between folic acid intake and colorectal cancer risk. Conversely, conventional treatment of existing tumours includes the use of folate antagonists. This suggests that the level of exposure to folate and its timing in relation to stage of tumorigenesis may be critical in determining outcomes. We hypothesised that folic acid depletion in utero and during early neonatal life may affect tumorigenesis in offspring. To investigate this hypothesis, female C57Bl6/J mice were randomised to a folic acid adequate (2 mg folic acid/kg diet) or folic acid depleted diet (0.4 mg folic acid/kg) from mating with Apc+/Min sires and throughout pregnancy and lactation. At weaning the Apc+/Min offspring were randomised to a folic acid adequate (2 mg folic acid/kg diet) or depleted (0.26 mg folic acid/kg diet) diet, creating four in utero/post-weaning dietary regimens. At 10 weeks post-weaning, mice were killed and the intestinal tumour number and size were recorded. Folic acid depletion during pregnancy and post-weaning reduced erythrocyte folate concentrations in offspring significantly. Folic acid depletion during pregnancy and lactation did not affect tumour multiplicity or size. However, female mice fed normal folic acid diets post-weaning had more, and larger, tumours when compared with depleted females and both depleted and adequate folic acid fed males. These data suggest that folate depletion post-weaning was protective against neoplasia in female Apc+/Min mice and highlights the need for further investigation of the optimal timing and dose of folic acid supplementation with regard to colorectal cancer risk.