Folate (vitamin B9) deficiency is a global health problem especially in developing countries where the major staple foods such as rice contain extremely low folates. Biofortification of rice could be an alternative complement way to fight folate deficiency. In this study, we evaluated the availability of the genes in each step of folate biosynthesis pathway for rice folate enhancement in the japonica variety kitaake genetic background. The first enzymes GTP cyclohydrolase I (GTPCHI) and aminodeoxychorismate synthase (ADCS) in the pterin and para-aminobenzoate branches resulted in significant increase in seed folate content, respectively (P < 0.01). Overexpression of two closely related enzymes dihydrofolate synthase (DHFS) and folypolyglutamate synthase (FPGS), which perform the first and further additions of glutamates, produced slightly increase in seed folate content separately. The GTPCHI transgene was combined with each of the other transgenes except ADCS to investigate the effects of gene stacking on seed folate accumulation. Seed folate contents in the gene-stacked plants were higher than the individual low-folate transgenic parents, but lower than the high-folate GTPCHI transgenic lines, pointing to an inadequate supply of para-aminobenzoic acid (PABA) precursor initiated by ADCS in constraining folate overproduction in gene-stacked plants.