Lipid droplets (LDs) function as intracellular storage depots of neutral lipids. Recently, we identified long-chain acyl-coenzyme A synthetase 3 (ACSL3) as a major LD-associated protein in the human hepatocyte cell line HuH7. In this study, we investigated whether droplet-associated ACSL is involved in lipid metabolism in LDs. Addition of oleic acid (OA) to culture medium was shown to enhance the intracellular accumulation of LDs in the cells, which was accompanied by an increase of droplet ACSL3. When LD-enriched cells induced by OA were further incubated without OA for 3 days, approximately 80% of LDs were retained in the cells. Conversely, cellular LD content was greatly decreased after the addition of an ACSL inhibitor, triacsin C. This was accompanied by a concomitant decrease of the droplet ACSL3. Incubation of isolated LD fractions with (14)C-labeled OA or palmitic acid resulted in [(14)C]acyl-CoA generation in vitro, indicating the presence of ACSL activity in LDs. The droplet ACSL activity varied according to the quantity of LDs in their emergence and disappearance in cells. Incubation of the LD fraction with [(14)C]oleoyl-CoA resulted in radioactive triacylglycerol and cholesteryl esters. These results suggest that LD ACSL activity is involved in local synthesis of neutral lipids and LD formation.