Galectin (Gal) constitute a family of carbohydrate-recognizing molecules ubiquitously expressed in mammals. In the immune system, they regulate many processes such as inflammation, adhesion, and apoptosis. Here, we report the expression in the spleen of the two same Gal-8 splice variants described previously in the thymus. Gal-8 was found to induce two separate biological activities on T lymphocytes: a robust naive CD4(+) T cell proliferation in the absence of antigen and notably, a costimulatory signal that synergized the cognate OVA peptide in DO11.10 mice transgenic for TCR(OVA). The antigen-independent proliferation induced by Gal-8 displayed increased expression of pro- and anti-inflammatory cytokines, thus suggesting the polyclonal expansion of Th1 and Th2 clones. The costimulatory effect on antigen-specific T cell activation was evidenced when the Gal and the peptide were assayed at doses suboptimal to induce T cell proliferation. By mass spectra analysis, several integrins and leukocyte surface markers, including CD45 isoforms, as well as other molecules specific to macrophages, neutrophils, and platelets, were identified as putative Gal-8 counter-receptors. Gal-8 triggered pZAP70 and pERK1/2. Moreover, pretreatment with specific inhibitors of CD45 phosphatase or ERK1/2 prevented its antigen-dependent and -independent T cell-proliferative activities. This seems to be associated with the agonistic binding to CD45, which lowers the activation threshold of the TCR signaling pathway. Taken together, our findings support a distinctive role for locally produced Gal-8 as an enhancer of otherwise borderline immune responses and also suggest that Gal-8 might fuel the reactivity at inflammatory foci.