Neurotensin (NT), a 13 amino-acid peptide, is predominantly released from enteroendocrine cells of the small bowel in response to fat ingestion. Free fatty acid receptors (FFARs) FFAR1 and FFAR4 regulate secretion of gut hormones and insulin. Here, we show that docosahexaenoic acid, a longchain fatty acid, has the most dramatic effect on NT release. FFAR1 agonists slightly stimulate and FFAR4 agonists dramatically stimulate and amplify NT secretion. Double knockdown of FFAR1 and FFAR4 decreases NT release, whereas overexpression of FFAR4, but not FFAR1, increases NT release. Administration of cpdA, an FFAR4 agonist, but not TAK-875, a selective FFAR1 agonist, increases plasma NT levels and further increases olive oil-stimulated plasma NT levels. Inhibition of MAPK kinase (MEK)/ERK1/2 decreased fatty acid-stimulated NT release but increased AMP-activated protein kinase (AMPK) phosphorylation. In contrast, inhibition of AMPK further increased NT secretion and ERK1/2 phosphorylation mediated by FFAR1 or FFAR4. Our results indicate that FFAR4 plays a more critical role than FFAR1 in mediation of fat-regulated NT release and in inhibitory crosstalk between MEK/ERK1/2 and AMPK in the control of NT release downstream of FFAR1 and FFAR4.