BACKGROUND: Glutathione is a valuable tri-peptide that is industrially produced by fermentation using the yeast Saccharomyces cerevisiae, and is widely used in the pharmaceutical, food, and cosmetic industries. It has been reported that addition of L-serine (L-Ser) is effective at increasing the intracellular glutathione content because L-Ser is the common precursor of L-cysteine (L-Cys) and glycine (Gly) which are substrates for glutathione biosynthesis. Therefore, we tried to enhance the L-Ser biosynthetic pathway in S. cerevisiae for improved glutathione production. RESULTS: The volumetric glutathione production of recombinant strains individually overexpressing SER2, SER1, SER3, and SER33 involved in L-Ser biosynthesis at 48 h cultivation was increased 1.3, 1.4, 1.9, and 1.9-fold, respectively, compared with that of the host GCI strain, which overexpresses genes involved in glutathione biosynthesis. We further examined simultaneous overexpression of SHM2 and/or CYS4 genes involved in Gly and L-Cys biosynthesis, respectively, using recombinant GCI strain overexpressing SER3 and SER33 as hosts. As a result, GCI overexpressing SER3, SHM2, and CYS4 showed the highest volumetric glutathione production (64.0 ± 4.9 mg/L) at 48 h cultivation, and this value is about 2.5-fold higher than that of the control strain. CONCLUSIONS: This study first revealed that engineering of L-Ser and Gly biosynthetic pathway are useful strategies for fermentative glutathione production by S. cerevisiase.