Abstract: OBJECTIVE To analyze the chemical constituents of the volatile oil from the fresh herbal medicine of Ferula ferulaeoides, and explore the anti-gastric cancer activity of D-limonene in vitro. METHODS GC-MS was used to analyze the chemical constituents of volatile oil of Ferula ferulaeoides; the MTT method was used to detect the proliferation inhibition effect of D-limonene on human gastric cancer cells AGS and SGC-7901; Hoechst 33258 fluorescent staining was used to observe the effect of D-limonene on the human gastric cancer cells AGS; the apoptosis of human gastric cancer cells AGS induced by D-limonene was detected by flow cytometry. RESULTS The results showed that α-pinene(6.76%), camphene(8.38%), α-myrcene(4.66%), 3-carene(4.84%), D-limonene(52.84%), bornyl acetate(5.24%), ect, were the main chemical constituents in the volatile oil of Ferula ferulaeoides. The MTT assay results showed that D-limonene had different degrees of proliferation inhibition effects on human gastric cancer cells AGS and SGC-7901, and showed a dose-dependent relationship. D-limonene had a strong proliferation inhibition effect on the human gastric cancer cell AGS, and its IC₅₀ was (0.87±0.05)mmol·L^-1. The cell nucleus of human gastric cancer cell lines AGS was stained by Hoechst 33258, and the blue fluorescence was stronger with the increase of drug concentration. The results of flow cytometry showed that D-limonene induced apoptosis of human gastric cancer cells AGS, and showed a dose-dependent relationship. CONCLUSION Monoterpenoids and sesquiterpenoids are main chemical constituents of the volatile oil of Ferula ferulaeoides. D-limonene is account for highest percentage of the total constituents. D-limonene has proliferation inhibition effect on human gastric cancer cells AGS and SGC-7901, the cytotoxicity activity of D-limonene on human gastric cancer cell AGS is strongest.