Intestinal epithelial barrier function is compromised in inflammatory bowel disease and barrier dysfunction contributes to disease progression. Extracellular nucleotides/nucleosides generated in gut inflammation may regulate barrier function through actions on diverse cell types. Enteric glia modulate extracellular purinergic signaling and exert pathophysiological effects on mucosal permeability. These glia may regulate inflammation with paracrine responses, theoretically mediated via adenosine 2B receptor (A_2BR) signaling. As the cell-specific roles of A_2BRs in models of colitis and barrier dysfunction are unclear, we studied glial A_2BRs in acute dextran sodium sulfate (DSS) colitis. We performed and validated conditional ablation of glial A_2BRs in Sox10^CreERT2+/−;Adora2b^f/f mice. Overt intestinal disease activity indices in DSS-colitis were comparable between Sox10^CreERT2+/–;Adora2b^f/f mice and littermate controls. However, ablating glial A_2BRs protected against barrier dysfunction following acute DSS-colitis. These benefits were associated with the normalization of tight junction protein expression and localization including claudin-1, claudin-8, and occludin. Glial A_2BR signaling increased levels of proinflammatory mediators in the colon and cell-intrinsic regulation of genes including Csf3, Cxcl1, Cxcl10, and Il6. Our studies show that glial A_2BR signaling exacerbates immune responses during DSS-colitis and that this adenosinergic cell-specific mechanism contributes to persistent gut epithelial barrier dysfunction.