Excessive hepatic lipid accumulation is a common phenomenon in cultured fish; however, its underlying mechanisms are poorly understood. Lipid droplet (LD)-related proteins play vital roles in LD accumulation. Herein, using a zebrafish liver cell line (ZFL), we show that LD accumulation is accompanied by differential expression of seven LD-annotated genes, among which the expression ofdehydrogenase/reductase(SDR family)member 3 a/b(dhrs3a/b) increased synchronously. RNAi-mediated knockdown ofdhrs3adelayed LD accumulation and downregulated the mRNA expression ofperoxisome proliferator-activated receptor gamma(pparg) in cells incubated with fatty acids. Notably, Dhrs3 catalyzed retinene to retinol, the content of which increased in LD-enriched cells. The addition of exogenous retinyl acetate maintained LD accumulation only in cells incubated in a lipid-rich medium. Correspondingly, exogenous retinyl acetate significantly increasedppargmRNA expression levels and altered the lipidome of the cells by increasing the phosphatidylcholine and triacylglycerol contents and decreasing the cardiolipin, phosphatidylinositol, and phosphatidylserine contents. Administration of LW6, an hypoxia-inducible factor 1α (HIF1α) inhibitor, reduced the size and number of LDs in ZFL cells and attenuatedhif1αa,hif1αb,dhrs3a, andppargmRNA expression levels. We propose that the Hif-1α/Dhrs3a pathway participates in LD accumulation in hepatocytes, which induces retinol formation and the Ppar-γ pathway.