Folates are vital one carbon donors and acceptors for a whole range of key biochemical reactions, including the biosynthesis of DNA building blocks. Plants use one carbon metabolism as a jack of all trades in their growth and development. Depletion of folates impedes root growth in Arabidopsis thaliana, but the mechanistic basis behind this function is still obscure. A global transcriptomic study hinted that folate depletion may cause misregulation of cell cycle progression. However, investigations on a direct connection thereof are scarce. We confirmed the effect of methotrexate (MTX), a folate biosynthesis inhibitor, on the expression of cell cycle genes. Subsequently, we determined the effect of MTX on root morphology and cell cycle progression through phase-specific cell cycle reporter analyses. Our study reveals that folate depletion affects the expression of cell cycle regulatory genes in roots, thereby suppressing cell cycle progression. We confirmed, through DNA labelling by EdU, that MTX treatment leads to arrest in the S phase of meristematic cells, likely due to the lack of DNA precursors. Further, we noted an accumulation of the A-type CYCA3
1 cyclin at the root tip, suggesting a possible link with the observed loss of apical dominance. Overall, our study shows that the restricted cell division and cell cycle progression is one of the reasons behind the loss of primary root growth upon folate depletion.