The characterization of mechanisms involved in the positive effects of probiotic bacteria in various pathophysiological conditions is a prerogative for their safe and efficient application in biomedicine. We have investigated the immunological effects of live bacteria-free supernatant collected from GABA-producing Lactobacillus brevis BGZLS10-17 on Concanavalin A-stimulated mesenteric lymph node cells (MLNC), an in vitro model of activated immune cells. We have shown that GABA containing and GABA-free supernatant of Lactobacillus brevis BGZLS10-17 have strong immunoregulatory effects on MLNC. Further, GABA produced by this strain exhibit additional inhibitory effects on proliferation, IFN-γ and IL-17 production by MLNC, and the expression of MHCII and CD80 on antigen presenting cells. At the other hand, GABA-containing supernatants displayed the strongest stimulatory effects on the expression of immunoregulatory molecules, such as Foxp3+, IL-10, TGF-β, CTLA4 and SIRP-α. By looking for the mechanisms of actions, we found that supernatants produced by BGZLS10-17 induce autophagy in different MLNC, such as CD4+ and CD8+ T lymphocytes, NK and NKT cells, as well as antigen presenting cells. Further, we showed that the stimulation of Foxp3+, IL-10 and TGF-β expression by BGZLS10-17 produced GABA is completely mediated by the induction of ATG5 dependent autophagy, and that other molecules in the supernatants display GABA-, ATG5-, Foxp3+-, IL-10- and TGF-β- independent, immunoregulatory effects.