Secretionof triacylglycerol-enrichedvery-low-density lipoproteins (VLDLs) fromthe liver is vital for maintaining plasma lipid homeostasis. However, the process of VLDL assembly and lipidation is not well characterized. Here, we observed that liver of Cideb null mice had higher levels of triacylglycerols accompanied by low level of VLDL secretion. Furthermore, VLDL particles secreted from hepatocytes of Cideb null mice have low levels of triacylglycerols but normal levels of apoB. We also observed that Cideb is localized to endoplasmic reticulum and lipid droplets. Importantly,wehave identified apoBas a Cideb-interacting protein. By infecting adenoviruses expressing various Cideb truncations into hepatocytes of Cideb null mice, we found that Cideb requires both its apoB-binding and lipid droplet association domains to restore the secretion of triacylglycerol-enriched VLDL particles. Our data suggest that Cideb promotes the formation of triacylglycerol-enriched VLDL particles and provides a molecular insight into VLDL lipidation and maturation in hepatocytes.