Background We previously showed that supernatants of Lactobacillus biofilms induced an anti-inflammatory response by affecting the secretion of macrophage-derived cytokines, which was abrogated upon immunodepletion of the stress protein GroEL. Methods We purified GroEL from L. reuteri and analysed its anti-inflammatory properties in vitro in human macrophages isolated from buffy coats, ex vivo in explants from human biopsies and in vivo in a mouse model of DSS induced intestinal inflammation. As a control, we used GroEL purified (LPS-free) from E. coli . Results We found that L. reuteri GroEL (but not E. coli GroEL) inhibited pro-inflammatory M1-like macrophages markers, and favored M2-like markers. Consequently, L. reuteri GroEL inhibited pro-inflammatory cytokines (TNFα, IL-1β, IFNγ) while favouring an anti-inflammatory secretome. In colon tissues from human biopsies, L. reuteri GroEL was also able to decrease markers of inflammation and apoptosis (caspase 3) induced by LPS. In mice, we found that rectal administration of L. reuteri GroEL (but not E. coli GroEL) inhibited all signs of haemorrhagic colitis induced by DSS including intestinal mucosa degradation, rectal bleeding and weight loss. It also decreased intestinal production of inflammatory cytokines (such as IFNγ) while increasing anti-inflammatory IL-10 and IL-13. These effects were suppressed when animals were immunodepleted in macrophages. From a mechanistic point of view, the effect of L. reuteri GroEL seemed to involve TLR4, since it was lost in TRL4 −/− mice, and the activation of a non-canonical TLR4 pathway. Conclusions L. reuteri GroEL, by affecting macrophage inflammatory features, deserves to be explored as an alternative to probiotics.