As the most important disulfide bond reducates of intracellular oxidordeuctase, thioredoxin (TRX) plays a crucial role in maintaining reducing state of intracellular proteins to normally perform their function. In this study, a cDNA of TRX-like protein gene from Apostichopus japonicus (denoted as AjTRX) was cloned and characterized. The full-length cDNA of AjTRXwas of 1870 bp, consisting of a 5′-UTR of 101 bp, a long 3′-UTR of 887 bp and a 882 bp open reading frame (ORF) encoding a 293 amino acids. The predicted molecular mass and the theoretical PI of the deduced amino acids of AjTRX were 32.3 kDa and 5.52, respectively. Phylogenetic trees showed that AjTRX had a closer evolution relationship with TRX from Strongylocentrotus purpuratus. AjTRX was found to be ubiquitously expressed in all examined tissues including longitudinal muscle, coelomocytes, tube feet, intestine, respiratory tree and body wall indicating a general role in physiological processes. Temporal expression pattern of AjTRX in coelomocytes showed that AjTRX reached two peak expression levels at 8 h and 48 h after Vibrio splendidus challenge with a 8.6 and 9.3-fold increase compared to their control groups, respectively. The recombinant AjTRX protein (rAjTRX) displayed obvious antioxidant activity in a dose-dependent manner, and the higher reducing activity was detected in 20 μM experimental group. All these results strongly suggested that AjTRX could play an important role as an antioxidant in a physiological context, and might be involved in the process of bacterial challenge.