Background: We previously reported that, among all the naturally occurring amino acids, l-valine is the most powerful luminal stimulator of glucagon-like peptide 1 (GLP-1) release from the upper part of the rat small intestine. This makes l-valine an interesting target for nutritional-based modulation of GLP-1 secretion. However, the molecular mechanism of l-valine-induced secretion remains unknown. Methods: We aimed to investigate the effect of orally given l-valine in mice and to identify the molecular details of l-valine stimulated GLP-1 release using the isolated perfused rat small intestine and GLUTag cells. In addition, the effect of l-valine on hormone secretion from the distal intestine was investigated using a perfused rat colon. Results: Orally given l-valine (1 g/kg) increased plasma levels of active GLP-1 comparably to orally given glucose (2 g/kg) in male mice, supporting that l-valine is a powerful stimulator of GLP-1 release in vivo (P > 0.05). Luminal l-valine (50 mM) strongly stimulated GLP-1 release from the perfused rat small intestine (P < 0.0001), and inhibition of voltage-gated Ca²⁺-channels with nifedipine (10 μM) inhibited the GLP-1 response (P < 0.01). Depletion of luminal Na⁺ did not affect l-valine-induced GLP-1 secretion (P > 0.05), suggesting that co-transport of l-valine and Na⁺ is not important for the depolarization necessary to activate the voltage-gated Ca²⁺-channels. Administration of the K_ATP-channel opener diazoxide (250 μM) completely blocked the l-valine induced GLP-1 response (P < 0.05), suggesting that l-valine induced depolarization arises from metabolism and opening of K_ATP-channels. Similar to the perfused rat small intestine, l-valine tended to stimulate peptide tyrosine-tyrosine (PYY) and GLP-1 release from the perfused rat colon. Conclusions: l-valine is a powerful stimulator of GLP-1 release in rodents. We propose that intracellular metabolism of l-valine leading to closure of K_ATP-channels and opening of voltage-gated Ca²⁺-channels are involved in l-valine induced GLP-1 secretion. (Figure presented.)