Previously we have shown that glutathione can protect Lactococcus lactis against oxidative stress and acid stress. In this study, we show that glutathione taken up by L. lactis SK11 can protect itself against osmotic stress. When exposed to 5 M NaCl, L. lactis SK11 cells containing glutathione exhibited a significantly improved survival compared to the control. Transmission electron microscope showed that the integrity of L. lactis SK11 cells containing glutathione maintained for at least 24 h, whereas cell autolysis of the control occurred within 2 h after exposure to this osmotic stress. Comparative proteomic analyses using SK11 cells containing or not-containing glutathione with or without exposure to osmotic stress were performed. The results revealed that 21 out of the 29 differentially expressed proteins are involved in metabolic pathways, mainly in sugar metabolism. Several glycolytic enzymes of L. lactis were significantly upregulated in the presence of glutathione, which might be the key for improving the general stress resistance of the host strain. Taken together with previous studies, we demonstrated that glutathione plays important protective roles in L. lactis against multiple environmental stresses, thus glutathione can be considered as a general protectant to improve the robustness and stability of dairy starter cultures.