UNLABELLED: Within cellular membranes, sphingomyelin is associated with cholesterol and this complex facilitates homeostatic regulation of membrane viscosity. Acute cholesterol depletion increases the synthesis of very-long-chain (VLC) sphingomyelin, but a link between lipid sensing and sphingolipid synthesis is lacking. Using sphingolipid metabolic flux analysis, we observed that VLC-ceramide, the precursor to VLC complex sphingolipids that are produced in the Golgi apparatus, was rapidly consumed after cholesterol depletion, while synthesis of long-chain sphingolipids was unaffected. Sphingolipid trafficking assays showed that cholesterol depletion enhances VLC-Ceramide trafficking from the endoplasmic reticulum to the Golgi apparatus. Changes in the sizes of coatomer II ER exit sites were correlated with increased VLC-Ceramide trafficking and concomitant increase in sphingomyelin. Depletion of Sec16A, a component of the COPII network, abolished VLC-SM synthesis. This study reveals ER-to-Golgi trafficking of VLC-Ceramide as a key regulatory node in organelle membrane homeostasis pathways. SUMMARY: In cellular membranes, sphingomyelin is associated with cholesterol. Metabolic flux analysis of sphingolipid metabolism showed that synthesis rate of sphingomyelin, but not ceramide, was increased after depletion of cholesterol due increased rate of COPII-dependent ER-to-Golgi transport of ceramide.