Zika Virus (ZIKV), which is associated with microcephaly and Guillain-Barré syndrome, brought a serious public health impact to most of Latin America countries in 2015 and 2016 during its endemic outbreak. Still, there is no effective antiviral drug or vaccine for ZIKV infection, addressing urgency to develop therapeutic solutions for ZIKV infection. Previously, using high throughput screening (HTS) technology, several potential hit molecules against ZIKV have been discovered, and Methotrexate (MTX) was identified as one of them. While MTX has been clinically used as a chemotherapy and anti-rheumatoid reagent, in this study, I investigated the mechanism of action of MTX against Zika virus (ZIKV) in vitro using Vero cells and human neural stem cells (hNSCs). In plaque assays, the antiviral effects of MTX against ZIKV were studied, showing a ten-fold decrease of the virus titer. To understand the antiviral mechanism of action of MTX, we focused on MTX’ target enzyme, dihydrofolate reductase (DHFR), and its pathways. To confirm that the antiviral effect of MTX is caused from antagonism of DHFR, a downstream metabolite of DHFR pathway, Leucovorin, was used as together with MTX as co-treatment for ZIKV-infected host cells. Surprisingly, addition of Leucovorin rescued the ZIKV replication during MTX treatment, supporting the idea that the antiviral effect of MTX might be facilitated through inhibition of DHFR. The antagonism of DHFR leads to a decrease of the de novo synthesis of purines and pyrimidines. Additional antiviral mechanism of MTX were studied using GAT (Glycine, Adenosine, and Thymidine) medium in which adenosine alone showed the rescue effect on ZIKV replication from MTX treatment.