Glucocorticoids are steroid hormones with important functions in development, immune regulation and glucose metabolism. The adrenal glands are the predominant source of glucocorticoids, however, there is increasing evidence for extra-adrenal glucocorticoid synthesis in thymus, brain, skin and vascular endothelium. We have recently identified intestinal epithelial cells as an important source of glucocorticoids, which regulate the activation of local intestinal immune cells. The molecular regulation of intestinal glucocorticoid synthesis is currently unexplored. In this study we investigated the transcriptional regulation of the steroidogenic enzymes P450ssc and 11-hydroxylase, and the production of corticosterone in the murine intestinal epithelial cell line mICcl2, and compared it to that in the adronocortical cell line Y1. Surprisingly, we observed a reciprocal stimulation pattern in these two cell lines. Elevation of intracellular cAMP induced the expression of steroidogenic enzymes in Y1 cells whereas it inhibited steroidogenesis in mICcl2 cells. In contrast, phorbol ester induced steroidogenic enzymes in intestinal epithelial cells, which was synergistically enhanced upon transfection of cells with the nuclear receptors Steroidogenic Factor-1 (SF-1, NR5A1) and Liver Receptor Homolog-1 (LRH-1, NR5A2). Finally, we observed that basal and LRH-1/phorbol ester-induced expression of steroidogenic enzymes in mICcl2 cells was inhibited by the antagonistic nuclear receptor Small Heterodimer Partner (SHP). We conclude that the molecular basis of glucocorticoid synthesis in intestinal epithelial cells is distinct from that in adrenal cells, most likely representing an adaptation to the local environment and different requirements.