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• Upstream has the highest prokaryotic alpha-diversity index. • Upstream had a larger clustered network in the < 63 μm fractions. • Varied sediment fractions during soil erosion altered the prokaryotic community. The transfer of particle-bound prokaryotes and substrates from slopes to rivers during soil erosion is a crucial dynamic process that greatly influences terrestrial biological and geochemical cycles. Particle size strongly affects the transport distance of suspended sediments and likely induces prokaryote and substrate variations along the course of the river. Previous studies have mainly focused on the spatial redistribution of soil prokaryotes, whereas the variation in prokaryotic diversity and community composition among different particle size-fractions in suspended sediments remain unclear. In this study, suspended sediments were collected at four plots (upstream, middle stream, downstream, and dam) along a river that runs through a valley. Sampled sediments were size-fractionated into > 63 μm and < 63 μm particles (referred to as small and large particle fractions hereafter), to identify the corresponding prokaryotic community composition, interactions, and functions. The richness index (Chao1 and observed species) and the diversity index (Shannon) of sediment-associated prokaryotic communities significantly decreased from upstream towards dam plots, except in the small particle fraction in downstream and dam plots. The highest β-diversity index value was found for the large particle fractions, while the geospatial contribution to β-diversity varied. The concentration of Proteobacteria in downstream (41.39 %) and dam (34.86 %) plots was lower than that in the upstream (47.65 %) and middle stream (45.90 %) plots. In contrast, Cyanobacteria (4.89 % and 6.75 %) and Verrucomicrobiota (5.98 % and 11.00 %) concentrations were greater in the downstream and dam plots, respectively, than those in the upstream (0.93 % and 2.43 %) and middle stream (3.37 % and 3.11 %) plots, respectively. Additionally, the concentrations of Verrucomicrobiota and Bacteroidota were significantly higher in the small particle fractions (2.26–8.12 % and 4.33–7.72 %) than those in the large particle fractions (2.03–5.94 % and 3.70–7.11 %). Compared to the other plots, upstream plots had a larger clustered network and a greater number of co-occurrences within the prokaryotic community in the bulk sediment and in the small particle fraction, whereas more complex interactions among prokaryotic communities were found in the large particle fraction from the dam plots. Soil organic carbon (SOC), total nitrogen (TN), and Olsen phosphorus were the three most influential factors, explaining 67.41 % of the variation in prokaryotic community. Proteobacteria and Myxococcota correlated negatively with SOC, TN, and Olsen P but positively with C = O. Conversely, Cyanobacteria and Verrucomicrobiota correlated positively with SOC, TN, and Olsen P but negatively with C = O. Functional groups linked to the biogeochemical cycling of carbon (methanol oxidation and methylotrophy) were found in high concentrations, whereas those linked to nitrogen (nitrogen and nitrate respiration) were found in low concentrations in upstream and middle stream plots. Reassembly of the sediment physicochemical characteristics among geospatial plots along the transport transect significantly altered the prokaryotic community composition and metabolic functional groups. [ABSTRACT FROM AUTHOR]