Sulfur (S) is one of the most redox-sensitive elements and has a marked impact on the geochemical cycling of biogenic elements in freshwater sediments. Current understanding of the speciation of sedimentary S, and of the processes regulating it, is insufficient. In this study, the speciation and spatial variations of S and iron (Fe) in sediments (soils) from Lake Hongfeng, one of the largest freshwater lakes in Southwest China, were investigated using X-ray absorption near-edge structure (XANES) spectroscopy and diffusive gradient in thin film technique (DGT). The results show that S in sediments and soils was composed of seven fractions in different electronic oxidation states (EOSs), including (i) reduced S (R-S, G1, EOS = -1), (ii) lowly oxidized S (LO-S, including G2-G5; EOS = 0, 0.5, 2, and 3.7), and (iii) highly oxidized S (HO-S, including G6 and G7; EOS = 5 and 6). Proportional differences of S speciation in sediments and soils indicated that HO-S is largely reduced to LO-S and R-S during depositional processes. The HO-S fraction decreased in the top surface sediments and then increased in the deeper layers, whereas the R-S fraction showed the opposite trend, suggesting that sulfate reduction and re-oxidation processes occurred in the sediments. High ratios of soluble Fe/S provided a favorable foundation for the reduction and burial of sedimentary S. The speciation and spatial variations of S in freshwater sediments are controlled by complex environmental factors, including terrigenous material discharges, water redox conditions, and porewater chemistry (such as for pH, Eh, and reactive Fe). Our study will help to deepen the understanding of the geochemical dynamics of S in the sediments of freshwater ecosystems.