Background <p>During the scent secretion period, adult male muskrats (<i>Ondatra zibethicus</i>) produce a musk-like fragrance similar to that of the forest musk deers (<i>Moschus berezovskii</i>). However, the underlying mechanism of scent secretion in muskrats were not well understood. Scent glands and testicular tissues from 2-year-old muskrats during non-scenting and scenting periods were analyzed using transcriptomics and metabolomics to identify key genes, metabolites, and pathways involved in scent secretion.</p> Results <p>Key findings include: (1) Significant differences were observed in the tissue structure of scent glands and testes between the two phases. (2) In the scent glands, fifteen candidate genes were identified that primarily regulate the formation of major musk components through steroid biosynthesis, unsaturated fatty acid (UFA) synthesis, glyceride metabolism, cholesterol metabolism, and aldosterone-regulated sodium reabsorption pathways. (3) In the testes, seven key genes were identified that regulate muskrat sex hormone synthesis and action, thereby controlling musk secretion via steroid hormone biosynthesis, GnRH signaling, estrogen signaling, prolactin signaling, cholesterol metabolism, and aldosterone synthesis and secretion pathways. (4) Transcriptomic and metabolomic association analysis revealed that cholesterol metabolism, UFA biosynthesis, and fatty acid metabolism pathways played key roles in regulating scent secretion activity. Meanwhile, during the scent-secreting period, the testes increase the level of phosphatidylethanol through genes <i>Itpr1</i>, <i>Plcb2</i>, and <i>Prkca</i>, influencing the transport and biosynthesis processes of fatty acids and cholesterol in the scent glands via GnRH signaling transduction and prolactin signaling transduction pathways. In addition, the testes regulate the expression of <i>Hsd17b3</i> and <i>Cyp3a</i> in the scent glands through genes <i>Cyp11a</i> and <i>Hsd11b2</i> via the steroid hormone biosynthesis pathway, promoting the synthesis of sterols and steroids from cholesterol in the scent glands.</p> Conclusions <p>This study discovered that the testes regulate the synthesis and transport of key components in muskrat musk within the scent glands through genes (<i>Itpr1</i>, <i>Plcb2</i>, and <i>Prkca</i>, <i>Hsd11b2</i>, and <i>Cyp11a</i>) via GnRH signaling transduction, prolactin signaling transduction, and steroid hormone biosynthesis pathways.</p>