The saturation of sugar beet rotations under different fertilizer application systems and long-term cultivation induces significant changes in soil properties, leading to decreases in humus content, mineral nitrogen, phosphorus, and potassium. The study was conducted in a stationary multifactorial experiment in grain-beet crop rotations: crop rotation, row-crop, and grain-row crop rotations with the application of 40 t/ha of manure under sugar beets + NPK 100:90:90 and a variant without fertilizers. The paper presents the results of monitoring changes in humus content during each rotation, reduction of humus reserves in the plow layer, and physicochemical and agrochemical soil indicators. In the variants without fertilizers, we observed 0.24–0.41% decline in humus content in all crop rotations during 3 rotations of ten-field crop rotations (30 years). Overall, there occurred 0.89–1.00% decrease over 50 years of anthropogenic influence, equivalent to 31.8–35.7 t/ha, or 23.1–26.1% of initial reserves per hectare. Despite application of 40 t/ha of manure + NPK 100:90:90 under sugar beets, humus loss was 27.5 t/ha in the row-crop rotation and 16.8 t/ha in the grain-row crop rotation. Fertilizer application led to increase in exchangeable and hydrolytic soil acidity. With the application of 6.7 t/ha of manure + NPK 53:42:42 per 1 ha of crop rotation area, there was a tendency towards increase in mineral nitrogen content, mobile phosphorus doubled to 280.1–302.8 mg/kg compared to the variant without fertilizers, and exchangeable potassium decreased regardless of the fertilization system, which was associated with its utilization by plants. Sugar-beet yield increased to 44.76 t/ha in the crop-rotation under the organo-mineral-fertilizer application system, exceeding the spring wheat rotation by 4.63 t/ha and the variants without fertilizers by 2.45–2.72 times. Therefore, the modern fertilizer application system under sugar beets did not ensure stabilization of humus content in the soil and increased its acidity. It is necessary to more broadly use cover crops in crop rotations, incorporate crop residues, and apply biological preparations to improve soil fertility.