Although mechanical tension from luminal distension is a primary regulator of gut motility, we reveal a parallel chemosensory pathway wherein long-chain unsaturated free fatty acids (LUFFAs) from dietary or enterobacterial sources directly modulate gastrointestinal motor function. Using ex vivo and in vivo contractility assays in human and murine intestinal tissues, we found that LUFFAs, particularly Omega-3 fatty acids, suppressed spontaneous contractions and delayed intestinal transit in a double bond-dependent manner. Mechanistically, selective activation of free fatty acid receptor 1/4 (FFAR1/4) on nitrergic myenteric ganglia triggered a rise in intracellular calcium and nitric oxide release, inducing smooth muscle relaxation independent of epithelial signaling. Genetic ablation of Ffar4 in enteric neurons or defect in enteric nitrergic ganglia abolished LUFFAs-mediated motility suppression and ameliorated colonic dysmotility induced by pathologically elevated LUFFAs levels. Our findings establish nitrergic ganglia as critical chemosensors translating dietary or enterobacterial lipid signals into gut motor responses.