Galectin-1 is a β-galactoside binding lectin with multiple immune functions in higher vertebrates. We report the characterization of two galectin-1 proteins from Atlantic cod, with emphasis on mucosal tissues. Tissue distribution of these two ≈ 14kDa galectin-1 proteins (Codgal1-1 and Codgal1-2) was ascertained by western blotting of one dimensional (1D) and two dimensional (2DE) gels. The two galectin-1 proteins were differentially localized in the mucosal tissues of cod. Codgal1-1 was predominantly localized in the basal cells of skin and this protein was present in all the early developmental stages examined, indicating a likely involvement in developmental processes. The two lectins were also localized in the adherent macrophage-like cells (MLC) from cod head kidney and results gathered indicate their possible secretion during Francisella noatunensis infection, suggesting that they are active components of immune defence. Lactose affinity chromatography coupled with gel filtration co-purified the two cod galectin-1 proteins, which hemagglutinated horse red blood cells in a lactose inhibitable manner. They also could bind and agglutinate both Gram-positive and Gram-negative bacteria. This study suggests multiple functional roles for galectin-1, especially in development and innate immune response of Atlantic cod.