Significance Folate deficiency is associated with multiple disorders in humans. Through the analysis of the fragile X syndrome locus ( FRAXA ) in immortalized human lymphocytes or fibroblasts, we demonstrate that FRAXA undergoes DNA synthesis in mitosis (MiDAS). We demonstrate that this process occurs via break-induced DNA replication and requires the SLX1/SLX4 endonuclease complex, the RAD51 recombinase and POLD3, a subunit of polymerase delta. We also demonstrate that other loci undergo MiDAS upon folate stress. This study reveals a function of human SLX1 in the maintenance of FRAXA stability and provides evidence that, in addition to FRAXA, MiDAS occurs at other loci following folate deprivation. These findings provide insight into the diverse and detrimental consequences of folate deficiency in human cells.