Abstract <p><i>Aspergillus niger</i> is a versatile biotechnological workhorse whose generalist lifestyle enables the utilization of a wide range of sugar compounds, including hexoses, pentoses, and polyols. Yet most predicted <i>A. niger</i> sugar transporters remain uncharacterized. Here, we characterized three glycerol transporters—GlpA, GlpB, and GlpC—from <i>A. niger</i>. We generated single and double <i>glp</i> deletion strains for physiological analysis, among which the Δ<i>glpA</i> strain showed a clear phenotype with significantly reduced glycerol consumption, indicating that GlpA is essential for glycerol uptake. Furthermore, both growth on glycerol and its uptake were abolished in the Δ<i>glpA</i>Δ<i>glpB</i> mutant, while the Δ<i>glpA</i>Δ<i>glpC</i> mutant showed no growth phenotype on solid medium but lacked glycerol consumption in liquid cultures. This suggests that GlpB plays a supportive role in glycerol uptake, whereas GlpC affects it indirectly. Nevertheless, heterologous expression of the <i>A. niger glp</i> genes in a sugar uptake deficient <i>Saccharomyces cerevisiae</i> strain showed that all three transporters could transport not only glycerol but also D-mannitol, D-sorbitol, and D-xylitol. In addition, GlpC transported the hexose sugars D-fructose, D-glucose, and D-mannose. These results demonstrate the broad specificities of the <i>A. niger</i> Glp transporters, highlighting the need for thorough physiological and functional characterization of filamentous fungal sugar transporters. Our findings advance the understanding of polyol and sugar uptake in <i>A. niger</i> and provide candidate transporters for strain engineering towards biotechnological upcycling of glycerol.</p> Key points <p>• <i>GlpA is the main glycerol transporter in A. niger under the studied conditions</i></p> <p>• <i>ΔglpAΔglpB and ΔglpAΔglpC double deletions abolished glycerol uptake in A. niger</i></p> <p>• <i>GlpA, GlpB, and GlpC have broad polyol specificity, with GlpC also uptaking hexoses</i></p>