Aims: To determine the role of the EpsA, EpsB, and EpsC proteins encoded at the 5'-end of the exopolysaccharide (EPS) gene cluster in regulation of EPS production in Lactococcus lactis. Methods and Results: Deletion and paralog-replacement mutants of epsABCD were used to determine the function of EpsA, EpsB and EpsC in EPS production and polymer chain length determination in L. lactis. EpsA and EpsB appeared to be essential for EPS biosynthesis in L. lactis, while deletion of the phosphatase (EpsC) only had a minor effect on the EPS production level. Determination of the phosphorylation state of EpsB and analysis of a C-terminally truncated EpsB variant indicate that EPS biosynthesis in L. lactis is driven by a nonphosphorylated form of EpsB. Conclusions: The data presented here show that in L. lactis, EPS production is under control of a phosphoregulatory system and that EPS biosynthesis correlates with an unphosphorylated EpsB. Significance and Impact of the Study: This study provides molecular understanding of polysaccharide production in L. lactis that could eventually enable novel approaches to control EPS production by lactic acid bacteria during industrial fermentation processes.