Abstract: Vegetables are important dietary sources of folate for human nutrition. The influence of different nitrogen doses and forms on changes in primary nitrogen metabolism, such as amino acid and protein synthesis, in plants is well established. However, the impacts of nitrate-N (NO₃^-)-to-ammonium-N (NH₄⁺) ratio on folate synthesis and accumulation in vegetables are unclear. This study used a hydroponic experiment with six different NO₃^-/ NH₄⁺ ratio treatments to investigate the effects of the integrated application of NO₃^- and NH₄⁺ on the folate constituents and contents of pakchoi (Brassica rapa subsp. Chinensis). The results indicated that an appropriate NO₃^- / NH₄⁺ ratio in nutrient solution could promote pakchoi growth and increase folate contents by increasing polyglutamylated 5-formyl-tetrahydrofolate (5-CHO-THF) and polyglutamylated 5-methyl-THF (5-CH3-THF). The activities of enzymes related to folate biosynthesis (except folylpolyglutamate synthase (FPGS)) were lower with an NH₄⁺-N supply at the same nitrogen concentration. The statistical results revealed a significant negative correlation between folate contents and 14 detected metabolites (including fructose and sucrose, glutamine (Gln) in the nitrogen assimilation, shikimate in the shikimate pathway, citrate, succinate, malate, α-oxoglutarate in the tricarboxylic acid cycle, and p-aminobenzoate (pABA) and 6-hydroxymethyldihydropterin pyrophosphate (HMDH-P2) in the folate biosynthesis pathway), implying that the enhancement of folates biosynthesis with NH₄⁺-N supply increased the consumption of the folate precursors and intermediate metabolites. Additionally, NH₄⁺-N supply could improve folate stability by increasing polyglutamylated folates and reducing γ-glutamyl hydrolase (GGH) activity; the latter could weaken folate deglutamylation. As the best growth and highest total folate content were obtained at the appropriate NO₃^-/ NH₄⁺ ratio, strategic selection of the NO₃^-/ NH₄⁺ ratio should be considered for the hydroponic cultivation of foliar vegetable crops.