Background: Guanylate cyclase-C (GC-C) signaling pathway via guanylin (Gn) and uroguanylin (Ugn) activation regulates intestinal fluid and electrolyte homeostasis. It is down-regulated in the patients of inflammatory bowel diseases (IBD). However, studies on the role of GC-C signaling in murine models of colitis are controversial. Here, we investigated the effect of GC-C on intestinal inflammation using Caco-2 cells. Methods: Caco-2 monolayers grown on Transwell filters were stimulated with IL-1 beta to model the inflammatory cells of intestine. The activity of GC-C signaling was regulated by transfection with Gn overexpression vector or GC-C shRNA plasmid. After different treatment with cells, the levels of Gn, Ugn, GC-C, paracellular permeability, superoxide dismutase (SOD), pro-inflammatory cytokines (IL-8 and TNF-alpha) and tight junction proteins (occludin, claudin-1 and ZO-1) were detected. Results: The expression of Gn, Ugn and GC-C was all significantly reduced after stimulation with IL-1 beta. Relative to the empty vector controls, IL-1 beta-treated cells transfected with Gn overexpression vector had significantly increased levels of Gn, GC-C, SOD, claudin-1 and ZO-1 as well as decreased levels of IL-8, TNF-alpha and permeability. Conversely, GC-C-silencing cells had significantly increased levels of IL-8, TNF-alpha and permeability as well as decreased levels of Gn, Ugn, GC-C, SOD, claudin-1 and ZO-1 induced by IL-1 beta compared with the corresponding empty plasmid controls. Conclusions: GC-C signaling pathway plays a protective role in the intestinal inflammatory injury and epithelial barrier function in Caco-2 cells. These observations further support the possible pathogenetic role of GC-C and clinical therapeutic potential of GC-C agonists in IBD.