Xylanases (EC 3.2.1.8) are enzymes that can hydrolyze the xylan backbone internally. Therefore, they are important for biomass breakdown and they are also often added in various biotechnological applications. In this study, the relationship between their substrate binding affinity and hydrolysis, on the one hand, and their movement over natural substrates, on the other hand, was investigated. Fluorescence recovery after photobleaching (FRAP) experiments using different Bacillus subtilis xylanase A (XBS) mutants were conducted on water-unextractable wheat flour arabinoxylan (WU-AX) and insoluble oat spelt xylan (OSX). To assess the importance of substrate hydrolysis, FRAP of a catalytically inactive mutant was compared to that of the wild-type enzyme. For the wild type enzyme substate binding and a complete recovery of fluorescence after photobleaching was observed on both substrates. For the inactive mutuant, however, substrate binding but no fluorescence recovery was observed on WU-AX, while very slow recovery was observed on OSX. Furthermore, the importance of substrate binding to a secondary xylan binding site (SBS) for enzyme mobility was studied by testing two mutants with a Modified SBS (N54W-N141Qand G56A-T183A-W185A) that showed different behavior on the tested substrates. On OSX, the two modified enzymes both showed higher mobility than the wild-type enzyme. On WU-AX, in contrast, the N54W-N141Q., mutant displayed a lower mobility than the wild-type enzyme, while the G56A-T183A-W185A mutant showed higher mobility. The results clearly demonstrate that both substrate hydrolysis and substrate targeting are key factors for XBS mobility.