DNA vaccines had been widely used against microbial infection in animals. The use of molecular adjuvants to improve the immunogenicity of DNA vaccines has been increasingly studied in recent years. MyD88 is one of the adapter molecules to activate the signaling cascades and produces inflammatory mediators, and its immunological role and adjuvant potential which had been proved in mammals were rarely reported in fish species. In this study, plasmid pcMyD88 was constructed and the capacity of MyD88 as molecular adjuvant was explored by co-injecting with a DNA vaccine encoding AcfA against Vibrio alginolyticus infection in orange spotted grouper. The results suggested that it needed at least 7 days to transported DNA vaccine pcacfA or molecular adjuvant pcMyD88 from the injected muscle to kidney and spleens and stimulate host's immune system for later protection. The co-injection of pcMyD88 with DNA vaccine pcacfA could increase significantly specific antibody levels and the expression levels of the immune-related genes including MHCIα, MHCIIα, CD4, CD8α, IL-1β and TNFα. Furthermore, pcMyD88 enhanced the immunoprotection of pcacfA against V. alginolyticus infection, with the significantly higher RPS of 83.3% in pcMyD88 + pcacfA group compared with that of pcacfA alone (73.3%) at challenging test of 10 weeks post vaccination. Together, these results clearly demonstrate that MyD88 is an effective adjuvant for the DNA vaccine pcacfA in orange spotted grouper.