Folic acid is recommended for rheumatoid arthritis patients to decrease the risk of methotrexate adverse effects. However, modification of immunosuppressive efficacy of methotrexate by folic acid is not precisely understood. We examined the influence of folic acid on the suppressive efficacy of methotrexate against mitogen-activated peripheral-blood mononuclear cells (PBMCs). PBMCs obtained from healthy subjects were cultured in the presence of methotrexate and/or folic acid under stimulation by concanavalin A for 96 h, and cell proliferation was estimated by MTT assay. Concentrations of seven inflammatory cytokines secreted into culture medium were measured by bead array procedures followed by flow cytometry. Percentages of CD4⁺, CD4⁺/CD25⁺, and CD4⁺/CD25⁺/FoxP3⁺ cells in lymphocytes were estimated by staining with specific antibodies. Methotrexate at 1-1000 ng/mL suppressed the proliferation of concanavalin A-activated PBMCs in a concentration-dependent manner, while 0.5-50 ng/mL of folic acid did not significantly disturb the methotrexate suppressive efficacy. Addition of folic acid at 24 h after the beginning of PBMC culture also had no significant effect on the methotrexate efficacy. Methotrexate tended to decrease the secretion of certain inflammatory cytokines, though the effects were not significant. Folic acid showed no significant influence on the methotrexate effects. Methotrexate at 100 ng/mL with or without folic acid significantly decreased the percentages of CD+4 cells in lymphocytes (p < 0.05), whereas the percentages of CD4⁺/CD25⁺ and CD4⁺/CD25⁺/FoxP3⁺ cells were not affected by methotrexate and/or folic acid. These results suggest that the suppressive efficacies of methotrexate on the activated PBMCs are not attenuated in the presence of folic acid at clinically used concentrations.