Understanding the molecular mechanism of host-pathogen interactions is the basis for drug design and vaccine development. The fine composition of mycolic acids (MA), the major constituents of Mycobacterium tuberculosis (Mtb) cell envelope, as well as other cell wall-associated lipids, contribute to determine the virulence of a given strain. However, endogenous receptors for mycolic acids on susceptible cells exposed to mycobacterial infections have not been fully identified. Here, we show that galectin-3, a multifunctional beta-galactoside binding lectin present mainly in the cytoplasm of inflammatory cells and also present on the cell surface, can recognize mycobacterial mycolic acids. MA can inhibit the lectin self-association but not its carbohydrate-binding abilities and can selectively interfere in the interaction of the lectin with its receptors on temperature-sensitive dendritic cell line, suggesting that galectin-3 could be involved in the recognition of trafficking mycolic acids and participate in their interaction with host cells.