Neutral sphingomyelinase (Smase) is a cell membrane-associated phospholipase that hydrolyzes sphingomyelin to phosphocholine and ceramide, a lipid second messenger involved in cell differentiation and/or apoptosis. We first evidenced that porcine cultured thyroid cells could express neutral Smase activity even if thyrotropin (TStH), an essential hormone in thyroid cell differentiation, was found to induce a 1.7-fold decrease in Smase activity. Triggering the ceramide pathway by exogenous addition of neutral bacterial Smase (0.1 U/mL for 48 h), which transiently increased ceramide level by fourfold, drastically modified thyroid cell morphology. The follicle-like structures generated by TSH were disrupted, and the Smase-induced cell spreading was accompanied by a parallel loss of cell ability to iodinate proteins as well as a decrease of the adenylate cyclase system response. These inhibitory effects have been reproduced using short-chain exogenous ceramide analogs (C2-ceramides). Overall these data showed that ceramides emerged as potential mediators of dedifferentiation in thyroid cells.