G protein-coupled receptors (GPCRs) have the potential to play a role as molecular sensors responsive to luminal dietary contents. Although such a role for GPCRs has been implicated in the intestinal response to protein hydrolysate, no GPCR directly involved in this process has previously been identified. In the present study, for the first time we identified GPR93 expression in enterocytes and demonstrated its activation in these cells by protein hydrolysate with EC₅₀ of 10.6 mg/ml as determined by the induction of intracellular free calcium. In enterocytes, GPR93 was synergistically activated by protein hydrolysate in combination with an agonist, oleoyl-L--lysophosphatidic acid (LPA), which activated the receptor in these enterocytes with EC₅₀ of 7.9 nM. The increased intracellular calcium by GPR93 activation was observed without the addition of a promiscuous G protein and was pertussis toxin sensitive, which suggests G_q and G_i mediated pathways. Activated GPR93 also induced pertussis toxin sensitive ERK1/2 phosphorylation. Both nuclear factor of activated T cells and 12-O-tetradecanoylphorbol 13-acetate responsive elements reporter activities were induced by protein hydrolysate in cells exogenously expressing GPR93. The peptidomimetic cefaclor by itself did not activate GPR93 but potentiated the protein hydrolysate response, and further amplified the synergistic enhancement of GPR93 activation by protein hydrolysate and LPA. These data suggest that physiologically the composition of stimuli might determine GPR93 activity or its sensitivity toward a given activator and suggests a new mechanism of the regulation of mucosal cell proliferation and differentiation, and hormonal secretion by dietary products in the lumen.