Signal transducer and activator of transcription 6 (STAT6) regulates transcriptional activation in response to interleukin-4 (IL-4)-induced tyrosine phosphorylation by direct interaction with coactivators. The CREB-binding protein and the nuclear coactivator 1 (NCoA-1), a member of the p160/steroid receptor coactivator family, bind independently to specific regions of STAT6 and act as coactivators. In this study we show that an LXXLL motif in the STAT6 transactivation domain mediates the interaction with NCoA-1. Peptides representing this motif as well as antibodies generated against this motif inhibited STAT6/NCoA-1 interaction in glutathione S-transferase pulldown assays. Peptides derived from the STAT6 transactivation domain adjacent to the LXXLL motif as well as antibodies against these peptides showed no inhibitory effect. Mutagenesis of the LXXLL motif eliminated the STAT6/NCoA-1 interaction in vitro and in vivo, supporting the specific role of this motif in NCoA-1 binding. Importantly, mutagenesis of the STAT-LXXLL motif strongly diminished the IL-4-regulated activation of the endogenous STAT6 target gene eotaxin-3. Taken together, these results indicate that the STAT6-LXXLL-binding motif mediates the interaction with NCoA-1 in transcriptional activation and represents a new potential drug target for the inhibition of the STAT6 transactivation function in allergic diseases.