Dendritic cells (DCs) are professional antigen-presenting cells that play a critical role in the immune system by directing the T lymphocyte response. Galectin-3, a protein found in DCs, is a structurally unique 946;-galactoside-binding animal lectin involved in cell homeostasis and immune regulation. In mouse models of allergic hypersensitivity and parasitic infection, galectin-3 deficient ( Lgals3-/- ) mice displayed differences in T helper type 1 (Th1) and type 2 (Th2) responses compared to wild-type mice. Accumulating evidence suggests that the altered T helper responses in Lgals3-/- mice may be attributed to DCs. Recently CD4+ Th17 cells have emerged as critical inducers of tissue inflammation in autoimmune disease and important mediators of host defense against extracellular and fungal pathogens. However, our current understanding of galectin-3 in regards to Th17 development is unclear. We sought to determine the role of galectin-3 in the induction of Th17 immunity through DCs. We demonstrate that galectin-3 in DCs negatively regulates Th17 polarization in response to fungal antigens both in vitro and in vivo. Upon activation of the fungal recognition receptor dectin-1, Lgals3 -/- DCs secreted higher levels of the Th17-associated cytokine IL-23 compared to Lgals3+/+ DCs and displayed higher levels of c-Rel, a transcription factor required for IL-23 expression. Furthermore, Lgals3-/- DCs had impaired levels of active Raf-1, a serine-threonine kinase that modulates DC cytokine expression by altering NF-954;B activation. Our findings suggest that therapeutic targeting of galectin-3 in DCs may be a means to modulate antifungal Th17 responses.Galectin-3 has also been identified as a protein component of DC-derived exosomes, small nanometer-sized vesicles secreted from cells that serve as messengers for intercellular communication. Using a combination of immunoblot, flow cytometry, and proteomic analysis to compare the protein compositions of Lgals3-/- and Lgals3 +/+ DC-derived exosomes, we demonstrate that galectin-3 may participate in protein sorting into multivesicular bodies (MVBs) and consequently affect protein sorting into exosomes. We specifically show that galectin-3 may negatively regulate expression of MHC-II and positively regulate expression of major vault protein into DC-derived exosomes. Consequently, the effect of galectin-3 on protein sorting into DC-derived exosomes may influence immune responses where exosomes are involved, such as in intercellular communication or T cell stimulation. Taken together, the data presented here represents novel roles for galectin-3 in the modulation of Th17 responses through regulation of DC cytokines and protein sorting into DC-derived exosomes. Considering the potent immunostimulatory capacity of DCs, elucidating roles for galectin-3 in DC functions and its subsequent effects on T cell activation may lead to the development of therapeutic vaccines for the manipulation of adaptive immunity.