The development of intestinal goblet cell hyperplasia/hypertrophy during nematode infection involves the Th2 cytokines IL-4 and IL-13 via STAT6 activation. This is thought to play an important role in host protective immunity against the infection. In this study we demonstrate that IL-4 and IL-13 up-regulate the specific goblet cell product trefoil factor-3 (TFF3) from the mucus-producing HT-29 CL.16E and HT-29 cells selected by adaptation to methotrexate. Up-regulation of TFF3 mRNA and protein levels occurred in a time- and dose-dependent fashion and was accompanied by up-regulation of the goblet cell product mucin 2 (MUC2). Addition of actinomycin D before IL-4/IL-13 stimulation led to decreases in TFF3 mRNA levels similar to those observed in controls without IL-4/IL-13. Furthermore, IL-4-mediated increased TFF3 transcription required de novo protein synthesis. Stable transfection of HT-29 CL.16E cells with a truncated dominant-negative form of STAT6 produced a cell line that was unresponsive to IL-4/IL-13. Although only one consensus STAT6 binding site is contained in the TFF3 gene, located in the intron 1, it did not operate as an enhancer in the context of an SV40 promoter/luciferase construct. Thus, STAT6 activation mediates a transcriptional enhancement of TFF3 by induction of de novo synthesized protein in goblet cells.