Fish are primary sources of the healthy n-3 long-chain polyunsaturated fatty acids (PUFA) for humans. This also applied historically to farmed fish, but the impact that nonmarine ingredients have on the nutritional quality of aquafeeds has prompted interest in the investigation of PUFA metabolism in farmed species of fish to optimize the endogenous production and retention of n-3 long-chain PUFA. The increasingly available genomic and transcriptomic data from fish, as well as the aforementioned interest in the contribution that farmed fish make to a healthy human diet, have greatly contributed to the understanding of the molecular mechanisms of PUFA metabolism in fish. This chapter focuses primarily on long-chain PUFA biosynthesis, although key processes of PUFA metabolism (digestion, absorption, and catabolism) that can affect tissue fatty acid compositions, and the key roles of PUFA in signaling pathways and the regulation of metabolism in fish, are also discussed.