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The present study investigated the effect of different dietary levels (0%, 0.25%, 0.5%, 1%, 2%) of Sophora alopecuroides (S. alopecuroides) on intramuscular fat (IMF) content in Ningxia Tan sheep. Candidate genes affecting IMF deposition were screened by comparing the RNA-Seq profiles of test (2% S. alopecuroides) and control (no S. alopecuroides) groups. The results showed that the intramuscular fat content of the diet supplemented with S. alopecuroides was significantly lower than that of the control group, and the intramuscular fat content decreased significantly with the increase of the dietary content of S. alopecuroides (P < 0.05). The highest IMF content of the control group (group A) was 4.21%, which was significantly higher than that of the other four experimental groups (B, C, D, E) (P < 0.05). Moreover, 66 genes were significantly upregulated and 202 genes were significantly downregulated in the test group compared to the control. Gene Ontology functional annotation revealed that the differentially expressed genes were closely related to plasma lipoprotein particle assembly, plasma lipoprotein particle organization, protein-lipid complex subunit organization, and other biological processes. KEGG pathway analysis showed that the differentially expressed genes belonged to 147 metabolic pathways. Finally, four differential genes which may be related to IMF deposition were screened out in PPAR signalling pathway, fatty acid biosynthesis, fatty acid degradation, and fatty acid metabolism. The quantitative analysis of four differential genes including ACSL3, PLIN2, ABCA1 and ANGPTL4 was carried out by RT-qPCR method, and the results were basically consistent with those of RNA-seq. Among them, the quantitative results of PLIN2 gene were slightly different from the sequencing results, but the difference was not significant, and they may be false positive. This study lays the foundation for understanding the molecular mechanism regulating mutton quality, and provides a theoretical basis for the study of S. alopecuroides as a feed additive to improve mutton meat quality and impact the gene function.