Chitosan is a polycationic amino-sugar polymer soluble in acidic pH. As a potential antifungal, it has been tested against several fungi. Its main mode of action is the permeabilization of cell membrane by the interaction with specific membrane sites. Ustilago maydis, an attractive fungal model used in biochemical and biotechnology research, is highly sensitive to chitosan, with extensive membrane destruction that results in cell death. Using the Golden Gate system, several mutant strains with deletions in monosaccharide transporters were obtained and tested against chitosan in order to know the implications of these membrane proteins in the sensitivity of the fungus against chitosan. Δum11514/03895 strain, a mutant with a deletion in a hypothetical high affinity glucose transporter, showed resistance to chitosan. Morphological characterization of the mutant displayed an apparent increase in mitochondrial content, but oxygen consumption as well as growth rate were not affected by the gene deletion. Alteration in cell wall surface was observed in the mutant strain. In contrast to wild type, the Δum11514/03895 strain showed integrity of cell wall and cell membrane in the presence of chitosan. The resistance against chitosan is likely associated to the modification of cell wall architecture and is not related to energy-depend process.