Cytokine production is the major regulators released from CD4+ T lymphocytes. Glycogen synthase kinase (GSK)-3β, a serine/threonine kinase, has been speculated for facilitating cytokine production probably by controlling several transcription factors. This study aimed to investigate the involvement of GSK-3β for cytokine production in CD4+ T lymphocytes. In an experimental model of CD4+ T lymphocyte activation, a combination of 12-O-tetradecanoylphorbol-13-acetate (TPA), used as an activator of protein kinase C (PKC), and ionomycin, used for calcium influx, so called T/I model, was utilized for this work. T/I treatment induced the production of cytokines IFN-γ, TNF-α, and IL-2 in human Jurkat T cells and primary CD4+ T lymphocytes. Treating cells with PKC inhibitor bisindolymaleimide or calcium chelator BAPTA blocked T/I-induced cytokine production. It is notable that treatment of GSK-3β inhibitor BIO and short hairpin RNA against GSK-3β decreased T/I-induced IFN-γ, TNF-α, and IL-2. Moreover, proline-rich tyrosine kinase 2 (Pyk2) inhibitor Tyrphostin A9 and calcineurin (PP2B) inhibitor cyclosporine A treatment also blocked cytokine production as well as GSK-3β activation in T/I-activated human CD4+ T lymphocytes. Activated GSK-3β regulated transcription factor T-bet, which was specifically and individually regulated for these cytokines under T/I stimulation. In a mice model, BIO treatment significantly inhibited T/I-induced mortality and the serum levels of cytokines. According to these results obtained from this work, targeting GSK-3β confers the therapeutic efficacy against CD4+ T cell activation and cytokine production.