Interleukin (IL)-1β is a potent mediator of innate immunity commonly upregulated in a broad spectrum of inflammatory diseases. When bound to its cell surface receptor, IL-1β initiates a signaling cascade that cooperatively induces the expression of canonical IL-1 target genes such as IL-8 and IL-6. Here, we present galectin-3 as a novel regulator of IL-1β responses in corneal keratinocytes. Using the SNAP-tag system and digitonin semipermeabilization, we show that recombinant exogenous galectin-3 binds to the plasma membrane of keratinocytes and is internalized into cytoplasmic compartments. We find that exogenous galectin-3, but not a dominant negative inhibitor of galectin-3 polymerization lacking the N-terminal domain, exacerbates the response to IL-1β by stimulating the secretion of inflammatory cytokines. The activity of galectin-3 could be reduced by a novel D-galactopyranoside derivative targeting the conserved galactoside-binding site of galectins and did not involve interaction with IL1R1 or the induction of endogenous IL-1β. Consistent with these observations, we demonstrate that siRNA-mediated suppression of endogenous galectin-3 expression is sufficient to impair the IL-1β-induced secretion of IL-8 and IL-6 in a p38 MAPK-independent manner. Collectively, our findings provide a novel role for galectin-3 as an amplifier of IL-1β responses during epithelial inflammation through an as yet unidentified mechanism. This article is protected by copyright. All rights reserved.