Royal jelly acid is a unique unsaturated fatty acid isolated from royal jelly. Recently, royal jelly acid was proposed to have potential therapeutic effects on hyperlipidemia. However, its effect on hyperlipidemia and the underlying molecular mechanism remains unclear. Therefore, in this study, we analyzed the mechanism of anti-hyperlipidemia of royal jelly acid through animal experiments and plasma metabolomics in conjunction with human network pharmacology and molecular docking analyses. We found that royal jelly acid could significantly decrease the serum lipid levels, ameliorate hepatic pathological injury, and reduce the level of oxidative stress in the experimental rats. A total of 41 key metabolites and 10 hub targets played key roles in the exertion of anti-hyperlipidemic effects, including tumor necrosis factor(TNF), insulin(INS) and epidermal growth factor receptor (EGFR). A total of 24 pathways, including tryptophan, citrate cycle, and arachidonic acid metabolisms, were identified as the key pathways involved in royal jelly acid-alleviated hyperlipidemia. The present findings provide new insights into the pathogenesis, diagnosis, and treatment targets of hyperlipidemia as well as contribute to the development and utilization of royal jelly acid related products.