MicroRNAs (miRNAs) have been recently shown to be important regulators of lipid metabolism. However, the mechanisms of miRNA-mediated regulation of long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis in vertebrates remain largely unknown. Herein, we for the first time addressed the role of miR-26a in LC-PUFA biosynthesis in the marine rabbitfish Siganus canaliculatus. The results showed that miR-26a was significantly down-regulated in liver of rabbitfish reared in seawater and in S. canaliculatus hepatocyte line (SCHL) incubated with the LC-PUFA precursor α-linolenic acid (ALA), suggesting that miR-26a may be involved in LC-PUFA biosynthesis due to its abundance being regulated by factors affecting LC-PUFA biosynthesis. Opposite patterns were observed in the expression of liver X receptor α (lxrα) and sterol regulatory element-binding protein-1 (srebp1), as well as the LC-PUFA biosynthesis related genes (Δ4 fads2, Δ6Δ5 fads2 and elovl5) in SCHL cells incubated with ALA. Luciferase reporter assays revealed rabbitfish lxrα as a target of miR-26a, and overexpression of miR-26a in SCHL cells markedly reduced protein levels of Lxrα, Srebp1 and Δ6Δ5 Fads2 induced by the agonist T0901317. Moreover, increasing endogenous Lxrα by knockdown of miR-26a facilitated Srebp1 activation and concomitant increased expression of genes involved in LC-PUFA biosynthesis, and consequently promoted LC-PUFA biosynthesis both in vitro and in vivo. These results indicate a critical role of miR-26a in regulating LC-PUFA biosynthesis through targeting the Lxrα-Srebp1 pathway and provide new insights into the regulatory network controlling LC-PUFA biosynthesis and accumulation in vertebrates.