Abstract: We previously identified two vertebrate peroxisome proliferator-activated receptor (PPAR) homologs, ScPPARa and ScPPARb, in Sinonovacula constricta. Here, we investigated their regulatory roles in the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFAs). Using bacteria expressing the respective dsRNA, we knocked down ScPPARa and ScPPARb transcription by about 50%. We analyzed the expression patterns of fatty acyl desaturases (Fads) and elongases (Elovls), along with fatty acid composition. ScPPARa-knockdown significantly downregulated Δ5 Fadb, Δ6 Fad, and Elovl4a but upregulated Elovl2/5. This phenomenon suppressed arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) levels in the muscle. In contrast, ScPPARb-knockdown reduced Δ5 Fadb, Elovl4a, and Elovlc, but increased the proportions of ARA, EPA, and DHA. Dual-luciferase assays combined with promoter truncation indicated that both ScPPARs could regulate Elovl2/5 and Elovl4a/b, while ScPPARb specifically modulated Elovlc and Δ6 Fad. Notably, changes in the expression patterns of several Fads and Elovls following ScPPARa or ScPPARb knockdown—unconfirmed by dual-luciferase assays—may be influenced by other transcription factors, such as sterol regulatory element binding proteins (SREBPs), which regulate LC-PUFA biosynthesis. Such alterations in gene transcription levels could be related to changes in fatty acid composition induced by a disruption of the PPAR signaling pathways. Overall, these findings reveal distinct regulatory roles of ScPPARa and ScPPARb in LC-PUFA biosynthesis. This is the first such report addressing the regulatory roles of PPARs in LC-PUFA biosynthesis in marine mollusks, providing foundational insights for optimizing LC-PUFA production in bivalves.