Secretion of various chemokines including Eotaxin-3/CCL26 results in the attraction of eosinophils to sites of allergic inflammation. IL-4/IL-13-induced activation of the Eotaxin-3/CCL26 gene in human dermal fibroblasts was shown to be a STAT6-dependent process mediated by a single STAT6 binding motif located upstream of the transcription initiation site. The suppressors of cytokine signaling 1–3 (SOCS 1–3) are members of a recently discovered family of proteins acting as negative regulators of cytokine signaling. We show here, that transfection of SOCS-1 and SOCS-3 but not SOCS-2 expression vectors inhibited IL-4/IL-13 induced secretion of Eotaxin-3/CCL26. Further, using Eotaxin-3/CCL26 promoter reporter gene constructs, we could show that, upon cotransfection of SOCS-1 and SOCS-3 expression vectors, IL-4 and IL-13 induced luciferase activity was strongly reduced. This effect was not seen when SOCS-2 was cotransfected. Further, EMSA studies with nuclear extracts prepared from IL-4/IL-13 induced HEK293 cells were conducted. The nuclear extracts of cells transfected with SOCS-1 or SOCS-3 did not form complexes with oligonucleotide probes corresponding to the STAT6 binding site in the Eotaxin-3/CCL26 promoter. In contrast, complex formation upon SOCS-2-transfection was comparable to mock-transfected cells. Further, the levels of phosphorylated STAT6 in IL-4 and IL-13 treated cells were markedly reduced when the cells had been transfected with SOCS-1 or SOCS-3, confirming the role of these negative regulators for the IL-4 and IL-13 induced activation of Eotaxin-3/CCL26 gene expression. The insertion of amino acid exchanges into the kinase inhibitory regions of SOCS-1 and SOCS-3 demonstrated a requirement of these domains for a proper inhibitory function.