The dopamine monoxygenase N‐terminal (DOMON) domain is found in extracellular proteins across several eukaryotic and prokaryotic taxa. It has been proposed that this domain binds to heme or sugar moieties. Here, we have analyzed the role of four highly conserved amino acids in the DOMON domain of the Drosophila melanogaster Knickkopf protein that is inserted into the apical plasma membrane and assists extracellular chitin organization. In principal, we generated Knickkopf versions with exchanged residues tryptophan299, methionine333, arginine401, or histidine437, and scored for the ability of the respective engineered protein to normalize the knickkopf mutant phenotype. Our results confirm the absolute necessity of tryptophan299, methionine333, and histidine437 for Knickkopf function and stability, the latter two being predicted to be critical for heme binding. In contrast, arginine401 is required for full efficiency of Knickkopf activity. Taken together, our genetic data support the prediction of these residues to mediate the function of Knickkopf during cuticle differentiation in insects. Hence, the DOMON domain is apparently an essential factor contributing to the construction of polysaccharide‐based extracellular matrices.