Purpose <p>To determine whether folate supplementation may reduce the incidence of Hirschsprung disease (HSCR) by regulating the methylation level of growth arrest-specific 5 (GAS5).</p> Methods <p>The m6A methylation deficiency model and folate supplementation model were created using the m⁶A inhibitor 3-Deazaadenosine (DAZ) and folate, respectively. The effects of folate on the morphology, mortality, and number of HuC/D positive neurons in zebrafish were observed. The methylation level and proliferation ability of mouse enteric neural crest stem cells (ENCSCs) were detected by m⁶A ELISA and EdU assay respectively. Human HSCR cases and control tissues were collected. The expression level of GAS5 in intestinal tissues was detected by qRT-PCR. M⁶A ELISA, actinomycin D, and MeRIP qRT-PCR were used to detect the effect of folate on m⁶A methylation levels, GAS5 stability, and the content of m⁶A-modified GAS5 in N2a cells. The RIP assay was used to detect the content of GAS5 bound to YTH N⁶-methyladenosine RNA binding protein F2 (YTHDF2) under different folate concentrations.</p> Results <p>Supplementation of folate can ameliorate the decreased ENCSC proliferation ability caused by low m⁶A methylation level, reduce embryonic mortality of zebrafish and increase the number of HuC/D positive neurons at 96 hpf. Furthermore, folate may reduce the expression of the demethylase alkB homologue 5 (ALKBH5), weaken ALKBH5's demethylation effect on GAS5, promote the degradation of GAS5 by YTHDF2, and subsequently reduce the expression of GAS5.</p> Conclusion <p>Additional folate supplementation during early embryonic development may serve as a strategy for preventing HSCR.</p>