Cholesterol-rich membrane domains, also called lipid rafts (LRs), are specialized membrane domains that provide a platform for intracellular signal transduction. Membrane proteins often cluster in LRs that further aggregate into larger platform-like structures that are enriched in ceramides and are called ceramide-rich platforms (CRPs). The role of CRPs in the regulation of intestinal epithelial functions remains unknown. Down-regulated in adenoma (DRA) is an intestinal Cl−/HCO3−antiporter that is enriched in LRs. However, little is known regarding the mechanisms involved in the regulation of DRA activity. The air–liquid interface (ALI) was created by removing apical media for a specified number of days; from 12–14 days post-confluency, Caco-2/BBe cells or a colonoid monolayer were grown as submerged cultures. Confocal imaging was used to examine the dimensions of membrane microdomains that contained DRA. DRA expression and activity were enhanced in Caco-2/BBe cells and human colonoids using an ALI culture method. ALI causes an increase in acid sphingomyelinase (ASMase) activity, an enzyme responsible for enhancing ceramide content in the plasma membrane. ALI cultures expressed a larger number of DRA-containing platforms with dimensions >2 µm compared to cells grown as submerged cultures. ASMase inhibitor, desipramine, disrupted CRPs and reduced the ALI-induced increase in DRA expression in the apical membrane. Exposing normal human colonoid monolayers to ALI increased the ASMase activity and enhanced the differentiation of colonoids along with basal and forskolin-stimulated DRA activities. ALI increases DRA activity and expression by increasing ASMase activity and platform formation in Caco-2/BBe cells and by enhancing the differentiation of colonoids.