Grazing prohibition is an effective measure in improving soil stability and ecological quality. However, only a limited number of studies have been published on the dominant factors that impact soil aggregate stability and their associated effects on nutrient distribution for different size soil aggregates under long-term grazing prohibition management. In this study, we investigated variation in soil aggregate stability and nutrient distribution characteristics in semiarid grassland sites under different grazing prohibition timeframes (0 years GP0, 11 years GP11, 26 years GP26, and 36 years GP36). Results showed that organic carbon (C) and total nitrogen (TN) concentrations in soil aggregates decreased at GP11 before progressively increasing and reaching its highest value at GP36, and the total phosphorus (TP) concentration did not change significantly. Most nutrients accumulated in macroaggregates (> 0.25 mm) under grazing prohibition, and the nutrient stoichiometry in soil aggregates increased after 26 years. Compared to the control (GP0), the mean weight diameter (MWD) value of the soil stability index increased at GP11 (21.7%) and decreased at GP26 (18.9%). Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) also showed that the proportion of stable organic C-related functional groups (i.e., alkene-C and aromatic-C) in macroaggregates were higher at GP11 and GP36 than at GP26. Furthermore, principal component analysis (PCA), partial least squares path modeling (PLS-PM), and the relative importance of regressors all showed that glomalin-related soil proteins (GRSP) and nutrients indirectly improved aggregate stability in semiarid grassland through their influence on the GRSP accumulation potential and nutrient stoichiometry. Generally, after 26 years grazing prohibition had a positive effect on soil aggregate stability and nutrient accumulation in the semiarid grassland sites investigated for this study. Results from this study provide a theoretical basis to select appropriate grazing prohibition timeframes under grassland management initiatives to optimize ecological quality measures in semiarid regions. Display omitted •Soil aggregates have more nutrients after 26 years grazing prohibition.•Soil aggregates' stability index MWD showed an order of 11 > 36 > 26 years.•GRSP potential and nutrient stoichiometry are the main factors controlling MWD.•High aggregate stability of semi-arid grasslands has more alkene-C and aromatic-C.•Grazing prohibition after 26 years favors the ecological quality of the grassland.