<jats:title>ABSTRACT</jats:title> <jats:p> dTDP-rhamnose is an important precursor of cell wall polysaccharides and rhamnose-containing exopolysaccharides (EPS) in <jats:italic>Lactococcus lactis</jats:italic> . We cloned the <jats:italic>rfbACBD</jats:italic> operon from <jats:italic>L. lactis</jats:italic> MG1363, which comprises four genes involved in dTDP-rhamnose biosynthesis. When expressed in <jats:italic>Escherichia coli</jats:italic> , the lactococcal <jats:italic>rfbACBD</jats:italic> genes could sustain heterologous production of the <jats:italic>Shigella flexneri</jats:italic> O antigen, providing evidence of their functionality. Overproduction of the RfbAC proteins in <jats:italic>L. lactis</jats:italic> resulted in doubled dTDP-rhamnose levels, indicating that the endogenous RfbAC activities control the intracellular dTDP-rhamnose biosynthesis rate. However, RfbAC overproduction did not affect rhamnose-containing B40-EPS production levels. A nisin-controlled conditional RfbBD mutant was unable to grow in media lacking the inducer nisin, indicating that the <jats:italic>rfb</jats:italic> genes have an essential role in <jats:italic>L. lactis</jats:italic> . Limitation of RfbBD activities resulted in the production of altered EPS. The monomeric sugar of the altered EPS consisted of glucose, galactose, and rhamnose at a molar ratio of 1:0.3:0.2, which is clearly different from the ratio in the native sugar. Biophysical analysis revealed a fourfold-greater molecular mass and a twofold-smaller radius of gyration for the altered EPS, indicating that these EPS are more flexible polymers with changed viscosifying properties. This is the first indication that enzyme activity at the level of central carbohydrate metabolism affects EPS composition.