Hyoscyamus niger L. is a medicinal plant which produces a class of jasmonate-responsive pharmaceutical secondary metabolites named tropane alkaloids. As a family of signaling phytohormones, jasmonates play significant roles in the biosynthesis of many plant secondary metabolites. In the jasmonate biosynthetic pathway of plants, allene oxide cyclase (AOC, EC 126.96.36.199) catalyzes the most important step. Here we cloned a cDNA from H. niger, named HnAOC (GenBank accession no.: AY708383), which was 1044 bp long, with a 747-bp open reading frame (ORF) encoding a polypeptide of 248 amino acid residues. Southern blot analysis indicated that it was a multicopy gene. RT-PCR analysis revealed that the expression of HnAOC was regulated by various stresses and elicitors, with methyl-jasmonate showing the most prominent inducement. The characterization of HnAOC would be helpful for improving the production of valuable secondary metabolites by regulating the biosynthesis of jasmonates.