Seasonal flooding-drought transformation process of lake sediments lead to changes of dissolved oxygen and redox conditions and the resultant generation of hydroxyl radical (HO). To date, information on HO formation and its regulators in seasonal lake sediments is largely unexplored. In this study, a total of nineteen sediments were collected from Lake Poyang, China, with the formation and mechanisms of HO during the oxygenation process exploring via the incubation experiments, Fe K-edge X-ray adsorption spectroscopy, ultrafiltration, and fluorescent spectroscopy. Results showed that the concentrations of HO generated ranged from 3.75 ± 1.13 to 271.8 ± 22.81 μmol kg, demonstrating high formation potential and obvious spatial heterogeneity. The yield of HO formed was positively correlated with the contents of Fe(II), sedimentary organic carbon, and dissolved organic carbon, showing a general contribution of these reduced substances to HO formation. Furthermore, application of Fe K-edge X-ray adsorption spectroscopy revealed the key species of sedimentary Fe-smectite for HO formation due to its high peroxidase-like activity. Besides inorganic Fe(II), the sedimentary dissolved organic matters (DOMs) represented an important regulator for HO formation, which contributed about 2-11% of the total HO generation. Moreover, the DOM-induced formation potential was found to be highly related to the molecular weight distribution that the low molecular weight- (LMW, <1 kDa) fraction exhibited higher HO formation potential than the bulk and high molecular weight- (HMW, 1 kDa-0.45 μm) counterparts. In addition, the omnipresent mineral Fe(II)-DOM interaction in sediment matrix exhibited another 2-6% of contribution to the total HO production. This study highlighted the importance of contents and species of Fe(II) and DOM in manipulating the HO yield, providing new insight into understanding the formation mechanisms of HO in the seasonal lake sediment.