Chondroitin sulfate synthase-1 (ChSy-1) is a crucial enzyme involved in the synthesis and elongation of chondroitin sulfate (CS), playing a vital role in combating bacterial and viral infections. The growing scale of aquaculture has led to the emergence of "rotting skin syndrome" caused mainly by Vibrio splendidus, posing a severe threat to the healthy cultivation of sea cucumbers. Our recent transcriptome study has found that ChSy-1 may have an essential immune function in protecting sea cucumbers from pathogenic infection; however, there has been no prior research on chondroitin sulfate synthase in echinoderms. In the present study, we obtained the full-length cDNA sequence of ChSy-1 using the RACE technique and analyzed its tissue expression characteristics in sea cucumbers using real-time fluorescence quantitative PCR. Furthermore, we also examined the expression characteristics of the body wall tissues under Vibrio vulnificus infiltration to investigate the possible physiological effects of AjChSy-1 on pathogenic infection. The study found that the AjChSy-1 gene has a full cDNA length of 2756 bp, with 5′-UTR length of 194 bp, 3′-UTR length of 228 bp, and ORF length of 2334 bp. The ORF encodes 777 amino acids, with a predicted molecular weight of the encoded protein of 89.1 kDa. The theoretical isoelectric point was 7.62, with a fat coefficient of 82.25 and an instability factor of 43.77. The protein sequence contains a type II transmembrane topology with 11 cysteine residues and four potential N-glycosylation sites. The three-dimensional spatial structure of the sea cucumbers AjChSy-1 protein is similar to that of the Holothuria leucospilota ChSy-1, with some differences from Homo sapiens. The sea cucumbers genome contains two copies of the AjChSy-1 gene, both of which are found on chromosome chr13. Gene structure analysis shows that both copies have two exons and one intron. Phylogenetic analysis showed that the protein encoded by AjChSy-1 in sea cucumbers was closely related to that of Holothuria leucospilota and Acanthaster planci, with similarities of 82.37% and 54.57%, respectively. It was more distantly related to that of Danio rerio, Homo sapiens, and other vertebrates. The protein encoded by AjChSy-1 contains structures such as a glycosyltransferase-conserved DXD motif, β3-glycosyltransferase motif, and β4-glycosyltransferase motif. Fluorescence quantitative PCR showed that the AjChSy-1 gene has a wide range of tissue expression characteristics, with the highest expression in the coelomocytes, followed by the body wall, gonads (male and female), and longitudinal muscles. The expression of AjChSy-1 in the body wall increased and then decreased with an increase in Vibrio splendidus stress duration. After 3 days of Vibrio splendidus imbibition, it increased sharply to 1.79 times of the control group and reached the highest transcript level of 3.12 times of the control group at 9 days. It then decreased starting from 12 days, and in diseased individuals, it decreased to 2.65 times of the control group. Upon analysis of the genes in both resistant and susceptible populations at the onset of the disease, it was revealed that the susceptible population had a markedly lower gene expression in their body wall compared to the resistant population (P＜0.05), with a difference of 11.4% in expression. According to the research findings, AjChSy-1 gene could potentially contribute to the immune protection against Vibrio splendidus in the sea cucumber Apostichopus japonicus. This study has also provided crucial insights into the regulatory mechanism of disease resistance and functional analysis of the AjChSy-1 gene in sea cucumbers.