Streptococcus iniae is a major fish pathogen that can also cause human bacteremia, cellulitis and meningitis. Screening for and identification of protective antigens plays an important role in developing therapies against S. iniae infections. In this study, we indicated that the -enolase of S. iniae was not only distributed in the cytoplasm and associated to cell walls, but was also secreted to the bacterial cell surface. The functional identity of the purified recombinant -enolase protein was verified by its ability to catalyze the conversion of 2-phosphoglycerate (2-PGE) to phosphoenolpyruvate (PEP), and both the recombinant and native proteins interacted with human plasminogen. The rabbit anti-rENO serum blockade assay shows that -enolase participates in S. iniae adhesion to and invasion of BHK-21 cells. In addition, the recombinant -enolase can confer effective protection against S. iniae infection in mice, which suggests that -enolase has potential as a vaccine candidate in mammals. We conclude that S. iniae -enolase is a moonlighting protein that also associates with the bacterial outer surface and functions as a protective antigen in mice.