Streptococcus agalactiae is an important pathogen associated with humans, animals, and fishes. Recently, it has spread globally, resulting in great economic losses in tilapia industries. The phosphotransferase system (PTS) is regarded as the "nervous system" of bacteria. The effect of the EIIB protein of cellobiose-PTS (cel-PTS) on S. agalactiae virulent strain virulence is limited, but it could potentially effect that of the attenuated strain; however, the specific mechanism is still unclear. Therefore, it is necessary to clarify the expression patterns of virulence-related genes regulated by the EIIB protein. In the previous study, the cel-EIIB gene deletion of the S. agalactiae virulent strain was constructed by homologous recombination technology. In this study, the cel-EIIB gene deletion of the S. agalactiae attenuated strain was obtained using a similar method. Zebrafish were infected with a S. agalactiae virulent strain, attenuated strain, and their cel-EIIB gene deleted strains, respectively. The results showed that S. agalactiae attenuated strain virulence was significantly stronger after cel-EIIB gene deletion, whereas S. agalactiae virulent strain virulence was only slightly reduced. It was confirmed by PCR detection that the deletion of cel-EIIB could cause cel-EIIA of the cel-PTS system, DltR and CiaH of the two-component signal transduction system (TCS), and sodA, cpsD, and cpsG of virulence genes showed opposite expression patterns in virulent and attenuated strains. In addition, the expression of RgfC, DltS, and CsrR of TCS and cspA and pavA of virulence genes were not affected in the mutant of the virulent strain, but up-regulated significantly in the mutant of the attenuated strain. It is speculated that the increased virulence of the S. agalactiae attenuated strain was related to the up-regulation of the expression of these virulence-related genes. To summarize, the EIIB protein may negatively regulate the virulence of attenuated strains by regulating the expression of the above virulence-related genes.