Crude elicitor of one endophytic fungi (belong to Cunninghamella sp., named AL4) induced multiple responses in Atractylodes lancea suspension cells, including rapid generation of nitric oxide (NO) and hydrogen peroxide(H2O2), sequentially followed by enhancement of essential oil production. Adding NO-specific scavenger 2-4-carboxyphenyl-4,4,5, 5-tetramethylimidazol ine-1-oxyl-3-oxide (cPTIO) and H2O2 scavenger catalase (CAT) could block elicitor-induced NO and H2O2 generation respectively, but could all partly block elicitor-induced essential oil biosynthesis. Adding NO-donor sodium nitroprusside (SNP) and H2O2 could all promote essential oil accumulation in A. lancea cells, but the effect of both was different. These results strongly suggested that NO and H2O2 may all act as signaling molecule to mediate AL4 elicitor promoting essential oil accumulation in suspension cells of A. lancea. Furthermore, adding cPTIO and CAT contemporarily could not completely inhibit essential oil accumulation induced by AL4 elicitor. This result suggested that AL4 elicitor could also promote essential oil accumulation in suspension cells of A. lancea by other means.