SOCS3, a negative regulator of cytokine signalling, is expressed in breast cancer cells where it can modify sensitivity and responsiveness to cytokines signalling through the JAK-STAT pathways. While it is widely accepted that SOCS3 expression is in itself regulated by STATs, we and others have shown that prostaglandins can also upregulate SOCS3 expression. Here we used T47D breast cancer cells treated with PGE₂ to examine this pathway. T47D cells responded to PGE₂ stimulation with a significant increase in SOCS3 mRNA that was independent of de-novo protein synthesis. PGE₂ stimulation resulted in STAT3 serine- and tyrosine-phosphorylation, although mutation of either of the two previously characterised STAT Response Elements (SREs) on the SOCS3 promoter did not affect SOCS3 promoter activation by PGE₂. In addition, overexpression of STAT3 wild type, constitutively active or dominant negative constructs did not affect PGE₂-induced SOCS3 promoter activation, indicating that STATs are unlikely mediators of this pathway in these cells. PGE₂ is a known activator of the cAMP/PKA pathway, and in T47D cells upregulation of SOCS3 mRNA by PGE₂ was abolished by pre-treatment with H89, a PKA inhibitor and increased by cAMP and Forskolin treatment. Consistent with this, PGE₂ treatment increased CREB serine-phosphorylation. However mutation of the AP1/CRE element on the promoter did not affect basal or PGE₂-stimulated activation, suggesting a role for cAMP/PKA that is independent of CREB binding. Mutation of the GC-rich region of the SOCS3 promoter, a putative Sp1/Sp3 binding site, abolished both basal and PGE₂-stimulated activation. Gel-shift assays showed increased complex formation following treatment and this was inhibited by the addition of an Sp1 antibody or pre-treatment with PKA inhibitor. ChIP assay verified Sp1 binding to the promoter in response to PGE₂. Sp1 overexpression increased SOCS3 promoter activation, and both basal and PGE₂-induced SOCS3 mRNA expression was prevented by mithramycin, an inhibitor of Sp1 DNA binding. Finally, a physiological role for PGE₂ was demonstrated with PGE₂ pre-treatment reducing LPS-induced STAT3 activation. Collectively this study details a novel mechanism of SOCS3 upregulation by PGE₂ in breast cancer cells which appears to be STAT-independent and involve Sp1 binding to the promoter. This process has possible implications for cytokine responsiveness and tumour progression.