Lipid droplets (LDs) are intracellular organelles that dynamically regulate lipids and energy homeostasis in the cell. LDs can grow through either local lipid synthesis or LDs fusion. However, how lipids involving in LD fusion for LD growth is largely unknown. Here we show that genetic mutation of acox-3 (acyl-CoA oxidase), maoc-1 (enoyl-CoA hydratase), dhs-28 (3-hydroxylacyl-CoA dehydrogenase), and daf-22 (3-ketoacyl-CoA thiolase), all involved in the peroxisomal β-oxidation pathway in C. elegans, led to rapid fusion of adjacent LDs to form giant LDs (gLDs). Mechanistically, we show dysfunction of peroxisomal β-oxidation results in the accumulation of long chain fatty acid-CoA (LCFA-CoAs) and phosphocholine (PC), which may activate the sterol regulatory element binding protein SBP-1/SREBP to promote gLD formation. Furthermore, we found inactivation of either FAT-2 (delta-12 desaturase) or FAT-3 and FAT-1 (delta-15 desaturase and delta-6 desaturase, respectively) to block the biosynthesis of polyunsaturated fatty acids with 3 or more double bonds (n≥3-PUFAs) fully repressed the formation of gLDs; in contrast, dietary supplementation of n≥3-PUFAs or PC bearing these PUFAs led to recovery of the formation of gLDs in peroxisomal β-oxidation-defective worms lacking PUFA biosynthesis. Thus, we conclude that n≥3-PUFAs, distinct from other well-known lipids and proteins, promote rapid LD fusion leading to LD growth.