Abstract: Chondroitin sulfate (CS) has been widely used as a dietary supplement in the food industry at present. However, there is incomplete absorption in the body, due to the large molecular weight, abundant hydrophilic groups, and low lipid solubility. As a result, CS exhibits low bioavailability (typically less than 13%), when taken orally. The bioavailability of oral CS formulations is closely linked to the molecular weight. Furthermore, the intact CS is still challenging to absorb in the digestive tract, where the low molecular weight CS (LMCS) can be absorbed through the intestine. LMCS can be expected to serve as the superior bioactivity and bioavailability, compared with undegraded CS. Therefore, the preparation of LMCS by degrading CS is of great significance for the further development and utilization of CS. There are several ways to prepare the LMCS so far, including free radical, enzyme, and acid degradation. Among them, the Fenton process is one type of free radical degradation with a commonly used and cost-effective reaction. It can be expected to degrade the CS by catalytic H₂O₂ while retaining the main polysaccharide structure. In this study, LMCS from different sources was prepared using the Fenton reaction, in order to investigate the potential degradation mechanism. The molecular weight, sulfate, and glyoxylate contents of chondroitin sulfate (BCS) of bovine origin were analyzed under different degradation conditions. The optimal CS degradation was determined using single-factor experiments. Subsequently, chondroitin sulfate derived from chicken (CCS), bovine (BCS), and shark (SCS) were reacted under optimal degradation conditions for 2, 4, 6, and 8 hours. The contents of sulfate and uronic acid, as well as the mono- and disaccharide composition and structure of the degradation products, were determined using Fourier transform infrared (FT-IR) spectroscopy and high-performance liquid chromatography (HPLC). For the first time, the potential mechanism of CS degradation by the Fenton reaction was analyzed from the perspective of molecular conformation and bond energy using molecular dynamics (MD) and standard density flooding theory (DFT). The results showed that the optimal conditions for CS degradation were a copper acetate concentration of 0.5 mmol, pH 7.5, and a reaction temperature of 45 ℃, respectively. The chemical composition of degradation products showed that the molecular weight of CS was positively correlated with the sulfate, glyoxylate, and GlcA content. The ratio of 4S/6S in the degraded CCS and BCS showed a decreasing trend with increasing degradation time, while there was no outstanding pattern for the degradation products of SCS. This infers that the GlcA residues of chondroitin-4sulfate (CSA) were preferentially attacked by free radicals in the Fenton degradation reaction. The FT-IR results of the degradation products indicated that the absorption peaks were related to the carboxyl derivatives formed by the free radical degradation of GlcA. MD experiments show that the conformational changes of CSA in H₂O occurred more frequently than those of non-sulfated chondroitin (CSO) and chondroitin-6sulfate (CSC), while the opposite result occurred in ·OH, where the RG and SASA values of CSA reached the minimum in ·OH. DFT calculations show that the C1-H bond of GlcA with radicals required a lower energy for the hydrogen abstraction reaction to occur, compared with GalNAc. Therefore, the C1-H of GlcA in CSA was more likely to undergo a hydrogen abstraction reaction with ·OH. These findings can provide promising guidance for the production of LMCS.