Changes in land use can significantly affect aggregate distribution and water stability of structural aggregates. This study was conducted in the Kolubara River Valley, Western Serbia, to determine the effects of land use changes on composition and water stability of aggregates in humus horizons (0–30 cm) of noncarbonated Gleyic Fluvisols. This study was conducted at nine sites, where each site contained two adjacent land uses of natural grassland and arable land which underwent crop rotation for >100 years. Soil samples were taken from depths of 0–10, 10–20 and 20–30 cm for each land use. When the grassland was converted into arable land, the content of the agronomically most valuable aggregates (0.25–10 mm) of cultivated soils for a depth of 0–30 cm was significantly reduced by 22–40%, while the percentage of cloddy aggregates (>10 mm) increased by 41–68%, compared to grassland. In addition, the long-term arable soil had significantly (p<0.05) lower aggregate stability, determined by wet sieving, than grassland. The lowest aggregate stability was found in aggregates > 3 mm. Their content is ≈ 2.3 times lower in arable soil (12.6%) than in grassland (28.6%) at a depth of 0–10 cm.In addition, meanweight diameters of dry and wet-stable aggregates and structure coefficient showed significant differences between land use at a depth of 0–30 cm. The results showed that the conversion of natural grassland to arable land in the lowland ecosystems of Western Serbia degraded aggregate distribution and stability.