The mucin Muc2 is the main component of the intestinal mucus layer and thus plays important roles in intestinal protection. Therefore, it is important to understand its regulation during goblet cell differentiation. Foxa1 and Foxa2 forkhead box transcription factors (TFs) participate in transcriptional programs governing intestinal cell differentiation. Using immunohistochemistry, we showed a spatio-temporal pattern of expression of both TFs in developing and adult mouse intestine and their expression in Muc2-expressing intestinal cells. Down-regulation of Foxa1 and Foxa2 by RNA interference in cultured intestinal cells decreased Muc2 mRNA level by half, and abolished Muc2 protein expression. Chromatin immunoprecipitation and gel shift assays showed that these two TFs directly bind to the Muc2 promoter. Co-transfection experiments indicated that both TFs activate the Muc2 promoter and that mutations of three Foxa cis-elements inhibit Muc2 transactivation. In conclusion, this work identifies Foxa1 and Foxa2 as important regulators of Muc2 expression in the intestine.