The homeobox transcription factor PROX1 is induced by high Wnt/ß-catenin activity in intestinal adenomas and colorectal cancer (CRC), where it promotes tumor progression. Here we report that in LGR5+ CRC cells, PROX1 suppresses the Notch pathway, which is essential for cell fate in intestinal stem cells. Pharmacological inhibition of Notch in ex vivo 3D organoid cultures from transgenic mouse intestinal adenoma models increased Prox1 expression and the number of PROX1-positive cells. Notch inhibition led to increased proliferation of the PROX1-positive CRC cells but did not affect their ability to give rise to PROX1-negative secretory cells. Conversely, PROX1 deletion increased Notch target gene expression and NOTCH1 promoter activity, indicating reciprocal regulation between PROX1 and the Notch pathway in CRC. PROX1 interacted with the nucleosome remodeling and deacetylase (NuRD) complex to suppress the Notch pathway. Thus, our data suggests that PROX1 and Notch suppress each other and that PROX1-mediated suppression of Notch mediates its stem cell function in CRC.