Objective To establish a female mouse model of high serum folate levels during peri-pregnancy by feeding a formula feed with high folate contents combined with serum folate testing, and to study the effects of high folate levels caused by folate supplementation during peri-pregnancy on offspring heart development. Methods According to the literatures, the formula feeds with normal folate content (2.0 mg/kg) and high folate content (10.0, 20.0, and 40.0 mg/kg) were designed. Thirty 6-week-old female BALB/c mice were randomly divided into 5 groups (6 in each group), 1 of which continued to be fed with common feed, and the other 4 groups were fed with the above 4 kinds of formula feeds. After 2 weeks of feeding, 4 groups of mice fed with formula feeds were divided into normal serum folate group (serum folate level＜ 2 000 pg/mL), high serum folate group (2 000 pg/mL≤serum folate level＜3 000 pg/mL), and ultra-high serum folate group (serum folate level≥3 000 pg/mL). After grouping, the female mice continued to be fed with the corresponding formula feeds and caged with male mice feeding with common feed. The gestational age was calculated as 0.5 d of pregnancy (E0.5) on the day when the vaginal plug was found. Cardiac tissue samples from fetal mice of different gestational ages were obtained, and cardiac development indicators at 10.5, 11.5 and 13.5 d of pregnancy (E10.5, E11.5, and E13.5) were detected by hematoxylin-eosin and immunohistochemical staining. Results With the increase of folate content in feed, the average serum folate levels of the female mice presented a gradually increasing trend, but there was no significant difference in serum folate level among different groups (P＝0.163). Compared to the normal and high serum folate groups, in the ultra-high serum folate group the cardiac tissue of fetal mice showed a loss of mesenchymal tissue (E10.5), decreases in the number and proliferation of myocardial cells (E10.5), thinner atrial and ventricular walls (E11.5, all P＜0.001), a decrease in the phosphorylated histone H3 positive rate (E11.5, both P＜0.001), a loss of dorsal mesenchymal protrusion (E13.5), structural abnormalities in right atrial appendage and ventricular outflow tract (E13.5), and ostium primum defect (E13.5). Conclusion It is feasible and effective to construct a female mouse model with high serum folate level by folate supplementation during peri-pregnancy combined with serum folate testing. The dramatic increase of serum folate levels in female mice during peri-pregnancy induced by folate supplementation may lead to cardiac dysplasia in offspring.