BACKGROUND & AIMS: In Crohn's disease, wrapping of mesenteric fat around the bowel wall, so-called "creeping fat," is highly associated with strictures. The strongest contributor to luminal narrowing in strictures is a thickening of the human intestinal muscularis propria (MP). We investigated creeping fat-derived factors and their effect on mechanisms of human intestinal MP smooth muscle cell (HIMC) hyperplasia. METHODS: Free fatty acids (FFAs) in creeping fat or noncreeping mesenteric fat organ cultures were measured via lipidomic mass spectrometry. Primary HIMCs were exposed to FFAs and cell proliferation was assessed. Intracellular FFA metabolism pathways and reactive oxygen species were functionally evaluated. Muscle thickness was investigated in dextran sodium sulfate colitis with small molecule inhibition of FFA transport and a novel fat deletion mouse model. RESULTS: Subserosal creeping fat is associated with a markedly thickened MP. Experimental deletion of mesenteric fat (FAT-ATTAC [fat apoptosis through targeted activation of caspase 8] mouse) reduced MP thickness. Human creeping fat-conditioned medium strongly up-regulated HIMC proliferation. Creeping fat released higher amounts of 5 long-chain FFAs, including palmitate. Inhibition of HIMC long-chain FFA metabolism or FFA uptake into mitochondria through carnitine palmitoyltransferase-1 reduced the palmitate-induced HIMC proliferation. Blockade of conversion of palmitate into phospholipids reduced HIMC proliferation. Prophylactic inhibition of carnitine palmitoyltransferase-1 in experimental dextran sodium sulfate colitis did not ameliorate inflammation, but reduced MP thickness. CONCLUSIONS: Creeping fat-released long-chain FFAs induce a selective proliferative response by HIMC. These results point to creeping fat as a novel contributor to stricture formation in Crohn's disease.