The aquatic zoonotic pathogen, Streptococcus iniae, represents a threat to the worldwide aquaculture industry and poses a risk to humans who handle raw fish. Because little is known about the mechanisms of S. iniae pathogenesis or virulence factors, we established a high-throughput system combining whole genome pyrosequencing and transposon mutagenesis that allowed us to identify virulence proteins, including Pdi, the polysaccharide deacetylase of S. iniae, that we describe here. Using bioinformatics tools, we determined a highly conserved signature motif in Pdi that is also conserved in the peptidoglycan deacetylase PgdA protein family. A pdi mutant was attenuated for virulence in the hybrid striped bass model and for survival in whole fish blood. Moreover, Pdi was found to promote bacterial resistance to lysozyme killing and the ability to adhere to and invade epithelial cells. On the other hand, there was no difference in the autolytic potential, resistance to oxidative killing, or resistance to cationic antimicrobial peptides between S. iniae wild type and pdi. In conclusion, we have demonstrated that pdi is involved in S. iniae adherence and invasion, lysozyme resistance, and survival in fish blood, and have shown that pdi plays a role in the pathogensis of S. iniae. Identification of Pdi and other S. iniae virulence proteins is a necessary initial step towards the development of appropriate preventive and therapeutic measures against diseases and economical losses caused by this pathogen.