Abstract: The calli of Cynanchum otophyllum were induced from the tender stems and young buds on MS medium with the supplement of 2.0 mg/L 2,4-D and 0.1 mg/L KIN. The result showed that 6,7-V medium was the optimal medium for callus growth. The tests using plant growth regulators with various concentrations showed that 6,7-V medium plus 2.0 mg/L 2,4-D and 0.3 mg/L KIN was the best combination for callus growth, whereas the production of secondary metabolites was higher on 6,7-V medium plus 1.0 mg/L 2,4-D and 0.1 mg/L KIN. The best period of culture was 27 d for callus growth, and 33 d for the maximal production of secondary metabolites. The isolation of nature products from the callus provided seven compounds including (1) fatty acid methyl ester named methyl 9,10,11-trihydroxy-12-octadecenoate and (2) daucosterol, (3) βsitosterol, (4) betulinic acid, (5) oleanolic acid, (6) hexadecanoic acid, (7) 9-octadecenoic acid. This is the first report of the isolation of poly-hydroxyl octadecenoic acid from a callus, and its proven biological function on the growth of plant cells was discussed.