To investigate the physiological roles of dietary arachidonic acid (ARA) in fish, a feeding trial with Japanese seabass was conducted, followed by a hepatic transcriptome assay. Six experimental diets differing basically in ARA level (0.05%, 0.22%, 0.37%, 0.60%, 1.38% and 2.32% of dry matter) were used in the feeding trial. Liver samples from fish fed diets with 0.05% and 0.37% ARA were subjected to transcriptomic assay, generating a total of 139 differently expressed unigenes, which were primarily enriched in lipid metabolism and cell cycle-related signaling pathways. Then, qRT-PCR validation on lipid metabolism and cell cycle-related genes as well as corresponding enzyme-linked immunosorbent assay of selected proteins were conducted with liver samples from all six groups. Moderated ARA levels reduced lipogenesis and stimulated β-oxidation concurrently, but high ARA levels seemed to affect lipid metabolism in complicated ways. Both gene expression and protein concentration of cell cycle-related proteins were decreased by moderate levels of dietary ARA. The lipid content and fatty acid composition in fish confirmed the transcription and protein concentration results related to lipid metabolism. In conclusion, moderate levels of dietary ARA (0.37% and 0.60%) reduced lipid accumulation and tended to inhibit cell cycle progression in the liver of Japanese seabass.