Maternal folate levels and polymorphisms in folate-related genes are known risk factors for neural tube defects (NTDs). SNPs in the mitochondrial folate gene MTHFD1L are associated with the risk of NTDs. We investigated whether different alleles of SNP rs7646 in the 3' UTR of MTHFD1L can be differentially regulated by microRNAs affecting MTHFD1L expression. We previously reported that miR-9 targets MTHFD1L and now we identify miR-197 as an additional miRNA regulator. Both of these miRNAs have predicted binding sites in the MTHFD1L 3' UTR in the region containing SNP rs7646. We have determined whether the alleles of SNP rs7646 (A/G) and miRNA expression levels affect miRNA binding preferences for the MTHFD1L 3' UTR and consequently MTHFD1L expression. Our results indicate that miR-9 and miR-197 specifically downregulate MTHFD1L levels in HEK293 and MCF-7 cells and that SNPrs7646 significantly affects miR-197 binding affinity to the MTHFD1L 3' UTR, causing more efficient posttranscriptional gene repression in the presence of the allele that is associated with increased risk of NTDs. These results reveal that the association of SNP rs7646 and NTD risk involves differences in microRNA regulation and, highlights the importance of genotype-dependent differential microRNA regulation in relation to human disease risk. © 2013 WILEY PERIODICALS, INC.