A number of studies have been demonstrated that arachidonate 5-lipoxygenase (ALOX-5) plays a role in regulating a range of physiological and pathological processes through the catalysis of leukotriene formation from arachidonic acid (ARA). The coding sequence of ALOX-5 from Apostichopus japonicus (Aj-ALOX-5) was successfully amplified, resulting in a 2028 bp ORF sequence that encodes 674 amino acids. A comparison of the amino acid sequence with those of other 5-lipoxygenases revealed that Aj-ALOX-5 has the N-terminal "PLAT domain" and C-terminal "lipoxygenase structural domain" characteristic of this enzyme family. The enzyme activity sites and Ca2⁺-binding sites exhibited high levels of conservation. The phylogenetic tree also indicated that Aj-ALOX-5 was closely related to starfish 5-lipoxygenase. The recombinant Aj-ALOX-5 (rAj-ALOX-5) was obtained through the exogenous expression of an engineered bacterium and purified using Ni2+-NTA. rAj-ALOX-5 was observed to catalyze ARA to produce 5-HPETE and LTA4, which indicated that the Aj-ALOX-5 protein belonged to the 5-lipoxygenase family. qRT-PCR demonstrated that Aj-ALOX-5 is widely distributed in tissues. Furthermore, the Aj-ALOX-5 mRNA and the production of 5-HETE were found to be significantly up-regulated in response to stress induced by Vibrio splendidus. Inhibition of Aj-ALOX-5 expression by the optimal caffeic acid resulted in a significant increase in mortality rates of sea cucumbers. Further investigation revealed that the production of 5-HPETE and NF-κB I was also significantly suppressed. It can be hypothesized that Aj-ALOX-5 plays an important role in the immune response of sea cucumbers by mediating the NF-κB I pathway.