Apolipoprotein (apo) C-III is a small protein (79 amino acids) and a component of triacylglycerol (TAG)-rich very low density lipoproteins (VLDL) and high density lipoproteins. We have unraveled a new intracellular role of apoCIII in promoting hepatic VLDL1 (Sf > 100) assembly/secretion under lipid-rich conditions. Feeding apoc3-null mice with a high fat diet for two weeks or palm oil gavage failed to stimulate VLDL1 production in vivo. Reconstitution of apoC-III expression using adenovirus encoding human apoC-III resulted in robust production of VLDL1 containing apoB-100 or apoB-48. The stimulatory effect of human apoC-III on the assembly and secretion of VLDL1 was recapitulated ex vivo in McA-RH7777 cells cultured in lipid-rich media. Metabolic labeling experiments showed that apoC-III plays a central role in (i) the formation of lumenal lipid droplets (LLD) rich in TAG, and (ii) promoting bulk TAG incorporation during VLDL1 assembly. Structure-function analysis of naturally occurring apoC-III variants (Ala23Thr and Lys58Glu) defined two functional domains that play respective roles in LLD formation and VLDL1 assembly. Unraveling the intracellular role of apoC-III in the atherogenic TAG-rich VLDL1 production provides new insights into the strong influence of the APOA5-A4-C3-A1 gene locus on plasma TAG concentrations and premature atherosclerosis.