Rotational woodlots with N2-fixing trees may be efficient agroforestry systems, allowing farmers to alternate agricultural and wood-energy production on the same area. However, their long-term effect on soil fertility is poorly understood. The aim of this study was to investigate the influence of successive phases of Acacia auriculiformis stands growing in rotation with crops on the chemical properties of sandy and very poor tropical soils. The study was conducted 22 years after the afforestation of humid herbaceous savannah in Mampu, Democratic Republic of Congo. The chemical properties of top soil (0–20 cm) from control savannah plots were compared with those from acacia plots that had undergone one, two or three rotations of acacia during the 22 year period. We found that the soil properties under non-harvested acacia stands in a 1st rotation and under acacia stands in a 2nd or 3rd rotation following charcoal production and maize and cassava cultivation were similar. Soils under all acacia stands had higher C, N and NO-3–N contents, but were more acidic, and had lower contents of exchangeable Ca, Mg, K and Na than the control savannah soils. Despite the increase in soil C and N, the sustainability of the acacia rotational agroforestry system after 22 years of practice is in question due to the steady decrease of soil cations, soil acidification and the risk of a decline in tree and crop productivity. To improve the nutrient balance and the sustainability of this system, different practices are recommended such as the debarking of tree stems before carbonization, the restitution of small branches and charcoal residues to the soil, and the supply of natural rock phosphate.