High water-level fluctuations (WLFs) usually lead to the periodic drying and rewetting of littoral sediment, proven to influence the transformation of various sediment contaminants. However, details regarding metal bioavailability variations remain unclear. In this study, oxidation–reduction status transition, metal fraction transformation, and the development of the acid volatile sulfide (AVS) and the simultaneously extracted metals (SEM) were investigated to assess the variations of metal bioavailability in the littoral anoxic sediment during the drying–rewetting process. A series of sediment cores were collected from the littoral area near the mouth of Nanfei River in Lake Chaohu, China, with half of them exposed to dark conditions with an overlying water cover (the control treatment). The other half was dried through the removal of overlying water and then exposed to air and sunlight (the drying treatment). Every 5 days, three of the dried sediment cores were rewetted for 3 days with lake water collected in situ. Various characteristics were analyzed for both the control and drying sediment cores. The drying and rewetting experiment was carried out for 30 days, and the bioavailability of metals was then evaluated thoroughly through data analyses. The periodic drying and rewetting process led to the oxidation of the anoxic sediment with a pronounced increase in oxygen penetration depth and redox potential, and a decrease in AVS concentrations in the drying treatment after 15 days of experimentation. Significant increases in the values of the ratio of SEM and AVS, the difference between SEM and AVS, and the organic carbon normalized difference between ΣSEM and AVS were retrieved for the drying treatment after process completion. The increase of metal bioavailability to aquatic organisms was consequently predicted, with the most significant concerns noted regarding Cd and Pb due to their transformation to less firmly bound fractions. The periodic drying and rewetting process could increase the bioavailability of metals in littoral anoxic sediment. Therefore, this process should be given more attention during future management of water level and remediation of littoral sediment in similar areas.