Liquid state fermentation is now a commonly used route to obtain triterpenoids from Antrodia cinnamomea, and linolenic acid can significantly promote triterpenoids synthesis, whereas its action mechanism has not been studied. Here, we comprehensively performed an investigation on the mechanism of linolenic acid to promote triterpenoids production in liquid‐state fermentation of A. cinnamomea. Results showed that the addition of linolenic acid increased the unsaturated fatty acid index, fluidity, and permeability in the cell membrane of A. cinnamomea mycelia, favored the absorption of nutrients in the medium by the mycelium, enhanced the material exchange inside and outside, and thus promoted mycelial growth and triterpenoids synthesis. Moreover, 767 significantly differentially expressed genes were detected by adding linolenic acid, including 212 upregulated genes and 555 downregulated genes. The upregulated genes were mainly enriched in metabolism, glycolytic pathway, TCA cycle, and pyruvate metabolism. It was seen that the addition of linolenic acid improved the cell metabolic activity and promoted the synthesis of secondary metabolites, proving that the addition of linolenic acid improved the metabolic viability of cells and promoted secondary metabolite synthesis.