In order to preliminarily illustrate the functional differences of phenoloxidases (POs) in Apostichopus japonicus, the full-length cDNAs of two POs (named as AjPOII and AjPOIII, respectively) were cloned from the coelomocytes of A. japonicus using 3′- and 5′-rapid amplification of cDNA ends method, and combined with the previously acquired full-length cDNA of a laccase-type PO from A. japonicus (Accession No. KF040052, named as AjPOI), the sequence structure and phylogenic status of POs from A. japonicus (AjPOs) were comparatively analyzed, and the transcriptional expression of AjPOs in different tissues, at different developmental stages and after different bacterial challenges was determined with quantitative real-time PCR method. Sequence analysis indicated AjPOII and AjPOIII were both laccase-type POs, coincident to the results of phylogenic analysis. Sequence analysis also showed that AjPOI had a transmembrane domain (J. Jiang et al., 2014), AjPOII contained a signal peptide, and AjPOIII possessed a signal peptide and a transmembrane domain, implying that three AjPOs might play different roles in immune and physiological processes. Transcriptional expression analysis showed that AjPOII and AjPOIII were most abundant in tube feet, while AjPOIhad the highest expression level in coelomocytes (J. Jiang et al., 2014), suggesting that AjPOI may be mainly involved in immune response, while AjPOII and AjPOIII are probably responsible for other physiological processes in addition to immune response. Besides, three AjPOs were determined to have different expression patterns during organism development and different spectrums of response against bacteria, which further indicated that there might be immune and physiological functional differentiation among three AjPOs.