Intestinal nutrient transport and sensing are of emerging interest in research on obesity and diabetes and as drug targets. Appropriate in vitro models are lacking that allow both, studies on transport processes as well as sensing and subsequent incretin hormone secretion including intracellular signaling. We here demonstrate that murine small-intestinal organoids are the first in vitro model system enabling concurrent investigations of nutrient and drug transport, sensing and incretin hormone secretion as well as fluorescent live-cell imaging of intracellular signaling processes. By generating organoid cultures from wild type mice and animals lacking different nutrient transporters, we show that organoids preserve the main phenotypic features and functional characteristics of the intestine. This turns them into the best in vitro model currently available and opens new avenues for basic as well as medical research.