Fractionations and mobility of heavy metals in sediment cores taken from the Suoxu River located in the Huaihe River watershed, China, were carried out and assessed. The results revealed that the total concentrations of Cd, Cr, Cu, Pb, and Zn reached up to 0.38, 70.2, 92.1, 35.9, and 227.0 mg kg−1 in sediment cores from the upstream, 0.19, 66.5, 27.4, 29.1, and 80.1 mg kg−1 from the midstream and 0.18, 49.4, 24.2, 28.5, and 64.8 mg kg−1 from downstream of the river, respectively. The concentrations of heavy metals near the surface of sediment cores from the upstream were significantly higher compared to the bottom and the downstream of the river. Significant positive correlations between total organic carbon and metals suggested that heavy metals tended to be adsorbed by organic material, especially in sediment cores from the upstream of the river possessing high concentrations of organic matter. In addition, the geoaccumulation index (Igeo) values of deep sediment samples were lower than those of surficial sediment samples and increased drastically from about 4 cm depth layer. Igeo values greater than 1 in the upper layer of sediments from the upstream of the river revealed that the upper stream of the Suoxu River was exposed to moderate pollution. The increase of metal concentrations was connected with the inflow of the contaminated water from industrial effluents and domestic sewage. Finally, the sequential extraction after the European Communities Bureau of Reference procedure indicated that Cd was primarily bound to acid extractable fractions with a percentage from 47.4 to 63.8%, 51.1 to 73.3%, and 47.5 to 71.9% in the three sediment profiles, respectively. The upstream of the river was exposed to toxicity of Cd for the significant mobility. Cu and Pb possessed potential bioavailability because of the association with reducible and oxidizable species and could be released and entered into the food chain, whereas Cr and Zn displayed weak mobility because of the connection with residual fractions, concluded from the largest proportions accounting for total concentrations.