Inflammatory mediators, including prostaglandins, cytokines, and chemokines, are strongly implicated in the mechanism of human labor, though their precise roles remain unknown. Here we demonstrate that interleukin 1 beta (IL-1beta) significantly increased the expression and release of interleukin-8 (CXCL8), monocyte chemotactic protein-1 (CCL2), and granulocyte macrophage colony-stimulating factor (CSF2) by primary human myometrial cells. However, this effect was repressed by prostaglandin E₂ (PGE₂). As PGE₂ can activate four distinct PGE₂ receptors (EP₁, EP₂, EP₃, and EP₄) to elicit various responses, we sought to define the EP receptor(s) responsible for this repression. Using selective EP receptor agonists and a selective EP₄ antagonist, we show that PGE₂ mediates the repression of IL-1beta-induced release of CXCL8, CCL2, and CSF2 via activation of the EP₂ and EP₄ receptors. The use of siRNA gene-specific knockdown further confirmed a role for both receptors. Real-time RT-PCR demonstrated that EP₂ was the most highly expressed of all four EP receptors at the mRNA level in human myometrial cells, and immunocytochemistry showed that EP₂ protein is abundantly present throughout the cells. Interestingly, PGE₂ does not appear to reduce mRNA expression of CXCL8, CCL2, and CSF2. Our results demonstrate that PGE₂ can elicit anti-inflammatory responses via activation of the EP₂ and EP₄ receptors in lower segment term pregnant human myometrial cells. Further elucidation of the EP receptor-mediated signaling pathways in the pregnant human uterus may be beneficial for optimizing the maintenance of pregnancy, induction of labor or indeed treatment of preterm labor.