The Three Gorges Reservoir (TGR) has formed the water-level-fluctuation zone (WLFZ) due to reservoir regulation. However, as a sensitive zone in reservoir, little is known about the geochemical process and ecotoxicological risk of arsenic (As) in WLFZ soils under the anti-seasonal flow regulation. Hence, the anthropogenic contamination, mobility and ecotoxicological risks of As in WLFZ soils of the TGR were comprehensively assessed using the geochemical baseline concentration (GBC), chemical fractions, diffusive gradients in thin films (DGT) and toxicity data. The As concentrations in WLFZ soils showed a trend of increasing at the early stage of water impoundment and then stabilizing in recent years, which presented a low ecological risk of As according to the assessment by pollution indices. Based on GBC calculations, the average anthropogenic contribution of As was 13.95 %, indicating a slight influence of human activities. The distribution of labile As measured by DGT in WLFZ soils was mainly controlled by the Fe/Mn oxides, pH and organic matter. The DGT-induced fluxes in soils (DIFS) model further implied that resupply of As to soil solution was partially sustained by the soil solid phase, in which the resupply capacity was low and limited by the adsorption and desorption kinetics. In addition, the DGT was combined with toxicity data to obtain the risk quotient (RQ) and probabilistic risk assessment. The RQ value was lower than 1, indicating a low toxicity risk in WLFZ soils. Furthermore, the As in WLFZ soils had a low probability (5.97E-3 % and 7.77E-2 % in the mainstream and tributary, respectively) of toxic effects toward the aquatic biota. This study provides a comprehensive evaluation for the mobility and toxicity risk of As in WLFZ soils, which is beneficial to the prevention and control of heavy metals pollution in the riparian soils of lakes and reservoirs.