Streptococcus agalactiae is the main pathogen of tilapia, causing huge economic losses to the tilapia aquaculture industry in China. To elucidate the role of the EII complexes of the cellobiose-phosphotransferase system (cel-PTS) in the regulation of S. agalactiae virulence, the △cel-EII mutants of a high virulence strain (THN0901) and a low virulence strain (TFJ0901) were constructed using homologous recombination. The results showed that after the deletion of cel-EII complexes, the biological characteristics of THN0901 and TFJ0901 were consistent, including their cellobiose metabolism. Their biofilm formation abilities were significantly reduced (P < 0.01), and their ability to adhere to tilapia brain cells and compete when mixed in vitro and in vivo were enhanced. The virulence and histopathological lesions caused by △cel-EII-TFJ0901 were significantly enhanced relative to those of TFJ0901, while △cel-EII-THN0901 showed no significant differences relative to THN0901. The histopathological lesions caused by △cel-EII-TFJ0901 tend to be serious, especially in the liver, spleen, and heart. The expression levels of manA and ascA/C of PTS, ciaR and dltR of two-component signal transduction systems (TCS), and pavA, bca and spb1 of virulence genes of △cel-EII-TFJ0901 were significantly upregulated compared with TFJ0901 (P < 0.01), whereas those of △cel-EII-THN0901 were significantly downregulated (P < 0.01), or somewhat downregulated, compared with THN0901. Taken together, cel-EII complexes may have a negative effect on the virulence of TFJ0901 by regulating the expression of virulence-related genes, but the negative regulation mechanism of the cel-EII complex has no obvious effect on the virulence of THN0901.