Our previous work has shown that T lymphocytes synthesize and secrete catecholamines (CAs), which regulate T cell function by paracrine/autocrine pathway. CD4+ T cells, a largest class of lymphocyte subsets accounting for 60% of peripheral T lymphocytes, function mainly through T helper (Th) cells. Th1 cells are mainly involved in inflammatory response by secretion of cytokines, such as interleukin (IL)-2, interferon-γ (IFN-γ) and tumor necrosis factor (TNF), while Th2 cells mainly mediate anti-inflammatory response by secretion of IL-4, IL-5 and IL-10. Th1/Th2 balance in differentiation and function is highly important for maintenance of immune homeostasis. Here, we explored the regulation of Th1/Th2 balance by CD4+ T cell-endogenous CAs to extend our knowledge of neuroimmunomodulation. CD4+ T cells were purified from the mesenteric lymph nodes of 4 to 6 week-old ICR mice by using CD4+ T-cell isolation kit. The purified T cells (1.25×106 cells/ml) were incubated for 48 h with concanavalin A (Con A, 5 μg/ml) to induce cell proliferation. To change production of CAs by the cells, we added alpha-methyl-p-tyrosine (α-MT), an inhibitor of tyrosine hydroxylase (TH) that is a rate-limiting enzyme for CA synthesis, or pargyline, an inhibitor of monoamine oxydase that degrades CAs, to the cultures 30 min prior to Con A. Additionally, recombinant TH miRNA expression vector (pcDNA/miR-TH) was transfected into Con A-activated CD4+ T cells using nucleofection technology to silence TH gene in these cells. Expression of TH and cytokines at gene and protein levels in Con A-activated CD4+ T cells was examined by real-time PCR and Western blot, respectively. Concentrations of CAs and cytokines in cultured cells and supernatants were measured by HPLC and flow cytometry, respectively. The exposure to α-MT (10−6 M) reduced contents of CAs including norepinephrine, epinephrine and dopamine both in CD4+ T cells and in supernatants. Simultaneously, α-MT upregulated T-bet, a Th1-specific transcription factor, and IFN-γ expression but downregulated GATA-3, a Th2-specific transcription factor, and IL-4 expression in Con A-activated CD4+ T cells. In contrast, pargyline (10−6 M) increased the three kinds of CAs, downregulated T-bet and IFN-γ, but upregulated GATA-3 and IL-4 in these lymphocytes. In support these findings, CD4+ T cells with TH RNAi expressed less TH mRNA and protein and synthesized less CAs than control cells with mock transfection. Importantly, the silencing of TH gene in Con A-stimulated CD4+ T cells elevated ratio of IFN-γ-producing cells to IL-4-producing cells, increased levels of IL-2, IFN-γ and TNF, but decreased levels of IL-4 and IL-5 in the culture supernatants. These data suggest that CAs synthesized and secreted by CD4+ T cells regulate differentiation and function of Th cells, with an effect facilitating the shift of Th1/Th2 balance toward Th2 polarization.