Abstract: The membrane proteins are critical to substrate protein secretion in VII secretion system existing in Streptococcus agalactiae. In this study, we constructed the membrane proteins knockout vectors with chloramphenicol tag including pSET4s-ΔessA, pSET4s-ΔessB and pSET4s-ΔessC, and after the homologous recombination, we screened the mutant S. agalactiae ΔessA, ΔessB and ΔessC via PCR assays. Based on the growth curve analysis, S. agalactiae ΔessA, ΔessB and ΔessC strains showed significantly slower growth process than the wild type strain (WT). ΔessA and ΔessB strains had significant difference in early stage of growth compared with WT strain (P<0.01). According to the secretion product analysis of ESAT6 protein, S. agalactiae ΔessA, ΔessB and ΔessC strains showed significantly lower ESAT6 product than WT (P<0.01). According to challenge test analysis, the deletion of essA, essB or essC gene significantly reduced virulence of S. agalactiae ΔessA, ΔessB and ΔessC compared with WT (P<0.01). The results suggest that essA, essB and essC proteins are important component membrane proteins of VII secretion system in S. agalactiae, and these genes deletions cause ESAT6 mRNA expression and virulence decline.