Oxylipins generated by the lipoxygenase (LOX) pathway play an important role in plant defense against biotic and abiotic stress. In chitosan-treated Adelostemma gracillimum seedlings, obvious accumulation of 9-LOX-derived oxylipins, namely 9,10,11-trihydroxy-12-octadecenoic acid, was detected. Using degenerate primers, a LOX-specific fragment putatively encoding LOX was obtained by RT-PCR, and a 2.9-kb full-length cDNA named AgLOX1 was isolated by RACE from chitosan-induced A. gracillimum seedlings. Genomic Southern analysis implied that there was only one copy of AgLOX1 in the A. gracillimum genome. AgLOX1 was expressed in Escherichia coli and the recombinant protein was partially purified. The enzyme converted linoleic and linolenic acids almost exclusively to their 9-hydroperoxides. AgLOX1 encoded a 9-lipoxygenase. Northern blot analysis indicated that chitosan-induced AgLOX1 transcript accumulation peaked at 8 h after initiation of treatment, whereas trihydroxy derivatives accumulation was highest at 24 h after elicitation. Results showed that chitosan-induced AgLOX1 encoded a 9-lipoxygenase potentially involved in the defense response through 9-LOX pathway leading to biosynthesis of antimicrobial compounds in A. gracillimum seedlings.