Streptococcus iniae (S. iniae) has become an important aquatic pathogen to cause serious streptococcosis outbreaks in cultured fish species, thus resulting in great economic losses in the worldwide aquaculture industry. Although vaccination has been so far an effective strategy to alleviate the threats of streptococcosis, antigenic variations displayed by different strains/isolates of S. iniae have brought about a severe challenge in the vaccine development. In the present study, three local S. iniae isolates, MML SI-007, MML SI-018 and MML SI-148, were first evaluated for their virulence in tilapia. Proteomics was then performed to analyze protein profiles between the highly virulent isolate (MML SI-007) and lowly virulent isolate (MML SI-018) in order to identify important virulence factors/antigens shared by different isolates. After proteomics, eleven proteins that displayed more than four times the differences in expression between MML SI-007 and MML SI-018 were identified. Two proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and superoxide dismutase (SOD), were selected to be vaccine candidates for respectively cloning their genes and expressing their recombinant proteins, rGAPDH and rSOD. After peritoneal vaccination in tilapia, mixed recombinant proteins (rGAPDH + rSOD) elicited significantly higher serum titers and lymphocyte proliferation than individual recombinant proteins (rGAPDH or rSOD). In addition, maximum production of IL-6 in the head kidney was detected in fish peritoneally vaccinated with mixed recombinant proteins. More importantly, 84% (21/25) of tilapia peritoneally vaccinated with mixed recombinant proteins survived at least four weeks after a lethal peritoneal challenge of heterologous S. iniae (MML SI-148). Taken together, our results have pointed out that both GAPDH and SOD identified via proteomics are crucial virulence factors that can be protective antigens for future development of an effective cross-protective subunit vaccine against S. iniae.