IIduronate-2-sulfatase (IDS) is a Ca2+-dependent enzyme belonging to the family of sulfatases that catalyzes the hydrolysis of sulphurylated glycosaminoglycans (GAGs), like dermatan and heparan sulphate. Its deficiency or modification leads to the accumulation of GAGs in the human body and to the occurrence of severe conditions, such as Hunter’s disease, or Mucopolysaccharidosis type II. Due to its involvement in this syndrome, it is of interest to understand the action mechanism of the enzyme to design new drugs for more efficient medical strategies. In the present work, we carried out a detailed multiscale modelling-based investigation, adopting molecular dynamics simulation (MDs) and QM/MM calculations to study the enzyme-dermatan sulfate interactions and to investigate the reaction mechanism of IDS. The analysis of molecular dynamics trajectories helped to shed light on the contribution of the individual residues of the active site in the recognition of dermatan sulfate. The role of FGly84 and His229, investigated in both neutral and protonated states, is highlighted for the binding of the substrate. QM/MM calculations demonstrated that the reaction mechanism is a multistep process occurring via a sulphurylation-desulphurylation mechanism where the FGly84 first attacks the sulphate group of the dermatan sulphate (DS), and later is desulphurylated.