Symplekin is a ubiquitously expressed protein involved in RNA polyadenylation and transcriptional regulation that localizes at tight junctions in epithelial cells. The association between symplekin and the Y-box transcription factor ZONAB activates proliferation in intestinal and kidney cells. We analyzed symplekin expression in human colonic crypts and investigated its function in differentiation.Expression of differentiation markers and transcription factors was assessed in HT29-Cl.16E cells that expressed inducible symplekin short hairpin RNA or were transfected with ZONAB small interfering RNAs. Intestines of AML1(Delta/Delta) mice were stained with alcian blue and analyzed for expression of AML1/Runx1, GAPDH, KLF-4, and Muc-2. Mobility shift and chromatin immunoprecipitation were used to detect AML1 and ZONAB/DbpA binding to promoter regions of the Krüppel-like factor 4 (KLF4) and acute myeloid leukemia-1 (AML1) genes, respectively.The gradient of nuclear symplekin expression decreased from the proliferative toward the differentiated compartment of colonic crypts; symplekin down-regulation promoted the differentiation of HT29-Cl.16E colorectal carcinoma cells into goblet cells. Down-regulation of symplekin or ZONAB/Dbpa induced de novo expression of the transcription factor AML1/Runx1, thereby increasing the expression of KLF4 and promoting goblet cell differentiation. Furthermore, increased AML1 expression was required for the induction of goblet cell differentiation after symplekin down-regulation. KLF4 expression and goblet cell numbers were reduced in the intestines of AML1(Delta/Delta) mice, confirming the role of AML1 as a promoter of intestinal differentiation in vivo.Symplekin cooperates with ZONAB to negatively regulate intestinal goblet cell differentiation, acting by repression of AML1 and KLF4.