An exopolysaccharide (EPS) producing strain of Lactococcus lactis subspecies cremoris was treated by pulsed electric field (PEF) while the strain was growing on the exponential phase and actively producing the EPS, in order to study how PEF treatment impacts on the biosynthesis of EPS. If a pulse with electric field of 8 kV/cm and pulse-length of 1 mu s was shot for 200 times by one-pass treatment, the EPS yield was increased for 32%. However, the same PEF treatment applied by circular treatment for 4h resulted in 94% increase versus the EPS yield by normal fermentation. The permeability of cells increaed only for 7-10% by the treatment, which suggested that moderate PEF conditions should have allowed enhancing the EPS production without heavily damaging the cells. The size exclusion chromatography showed that the EPS peak shifted to-lower-molecular weight by the PEF treatment, although the chemical compositions of EPS with and without PEF treatment were almost identical. R-Galactosidase activity and polyisoprenoid-linked intermediates of EPS biosynthesis were analyzed as a clue to postulate the mechanism. The study suggested that PEF treatment can be a potential engineering technique to enhance EPS production by L. lactis subsp. cremoris. (C) 2016 Elsevier Ltd. All rights reserved.