Cyanobacterial blooms are a major environmental problem in eutrophic reservoirs in China. Algal cells can migrate to the sediment surface in winter and maintain biological activity, which could further affect the cycling process of sediment phosphorus (P) and iron (Fe). In this study, a pilot simulation experiment was conducted to investigate the effect of overwintering cyanobacteria (Owc) on P and Fe regeneration across the sediment-water interface (SWI). Owc esterase activity ranged from 16.4 to 26.6 nmol (FDA)/(L·h), with a fluctuating increasing trend within the incubation time. Compared with the control (no Owc), Owc treatment increased the redox potential value (Eh) at the SWI but slightly decreased the pH during the first stage of this experiment (0–24 d); however, the Eh at the SWI under Owc treatment decreased to 50.9 % of that of the control on day 90. The Fe(II) could rapidly oxidized to Fe (oxyhydro)oxides and combine with phosphate in high Eh environments, and Owc inhibited P and Fe release at the SWI within 24 days; however, the continuous decrease in Eh resulted in the reduction of Fe(III). Thus, the Fe concentration measured via diffusive gradients in thin films in the Owc-treated interstitial water gradually increased to 1.92 times that of the control, promoting the release of Fe and P across the SWI. For 13 days after Owc addition, the amount of mobile P in the sediment was significantly higher than that in the control, and it gradually decreased from day 24 to 90, with the lowest being approximately 74.1 % of the amount in the control. The reactive Fe concentration in the sediment showed a similar variation trend. These results indicate that mobile P and reactive Fe in the sediment could be the main sources of regeneration across the SWI in the presence of Owc.