1321N1 human astrocytoma cells express thromboxane A₂ (TXA₂) receptors (TP). However, physiological consequences of TXA₂ signaling in glial cells remain unclear. Herein, we show that TXA₂ promotes interleukin-6 (IL-6) biosynthesis in glial cells. A TP agonist, 9,11-dideoxy-9α,11α-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U46619), enhanced IL-6 production in both 1321N1 cells and cultured mouse astrocytes. It has been shown that IL-6 gene expression is regulated by various transcription factors. Among them, we found a significant increase in cyclic AMP-response element-binding protein (CREB) activity with its phosphorylation at Ser133 by U46619 in 1321N1 cells. Although U46619 increased IL-6 promoter activity, a mutation at cyclic AMP-response element (CRE) on the promoter clearly suppressed the effect, suggesting that CRE is involved in U46619-induced IL-6 expression. Furthermore, both CREB and IL-6 promoter activities were suppressed by SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole], a p38 mitogen-activated protein kinase (MAPK) inhibitor, and H89 [N-[2-(4-bromocinnamylamino)-ethyl]-5-isoquinoline], a protein kinase A (PKA) inhibitor, indicating involvements of p38 MAPK and PKA in CREB activation and IL-6 expression. To determine which G-proteins are implicated in the U46619-induced IL-6 synthesis, the interfering mutants of Gα_q, Gα₁₂, or Gα₁₃ by were overexpressed in 1321N1 cells adenoviral approach. It is noteworthy that the Gα_q or Gα₁₃ mutant blocked the IL-6 production by U46619. The constitutively active mutant of Gα_q, Gα₁₂, or Gα₁₃ enhanced IL-6 production, indicating that Gα_q and Gα₁₃ were involved in U46619-induced IL-6 production. In conclusion, TXA₂ enhances the IL-6 biosynthesis via the PKA p38 MAPK/CREB pathway in 1321N1 cells. IL-6 induction depends on Gα_q and Gα₁₃ as well. This is the first report showing TP-mediated IL-6 production in glial cells.