The genes of the non-phosphorylative L-rhamnose catabolic pathway have been identified for several yeast species. In Pichia stipitis, all L-rhamnose pathway genes are organized in a cluster, which is conserved in Aspergillus niger, except for the lra-4 ortholog (lraD). The A. niger cluster also contains the gene encoding the L-rhamnose responsive transcription factor (RhaR) that has been shown to control the expression of genes involved in L-rhamnose release and catabolism. In this paper, we confirmed the function of the first three putative L-rhamnose utilisation genes from A. niger through gene deletion. We explored the identity of the inducer of the pathway regulator (RhaR) through expression analysis of the deletion mutants grown in transfer experiments to L-rhamnose and L-rhamnonate. Reduced expression of L-rhamnose-induced genes on L-rhamnose in lraA and lraB deletion strains, but not on L-rhamnonate (the product of LraB), demonstrate that the inducer of the pathway is of L-rhamnonate or a compound downstream of it. Reduced expression of these genes in the lraC deletion strain on L-rhamnonate show that it is in fact a downstream product of L-rhamnonate. This work showed that the inducer of RhaR is beyond L-rhamnonate dehydratase (LraC) and is likely to be the 2-keto-3-L-deoxyrhamnonate. Overall design: This study aim to characterize in vivo the genes involved in the rhamnose metabolism and the regulator in Aspergillus niger by studing the gene expression values of the mutants and the reference strains. Mutant strains of the putative rhamnose metabolic and regulatory genes (lraA, lraB, lraC, rhaR) and reference strain were grown on L-rhamnose. Samples were taken after 2 hours of incubation.