A hairy root culture system of Calotropis gigantea was established and effects of mechanical wounding (MW) and elicitors [methyl jasmonate (MJ), yeast extract (YE) and chitosan (CS)] on cardenolide production were investigated. All treatments stimulated the production of cardenolide in hairy root cultures of C. gigantea. CS was the most effective elicitor, followed by MJ. YE and MW also improved cardenolide yield in individual treatments. The highest cardenolide yield (1,050 +/- 55 mg/l) was obtained after adding 50 mg CS/l for 20 days, which was 2.7-fold higher than the control.