Glomalin-related soil protein (GRSP) are a glycoprotein mainly produced by arbuscular mycorrhizal (AM) fungi. GRSP are deposited in the soil after being released from AM fungal hyphae, and they are believed to improve soil health and carbon (C) storage. However, it is unclear how fertilizer and mycorrhiza suppression affect the content of easily extractable (EE-GRSP) and total glomalin-related protein (T-GRSP) in arid grassland soil. We conducted a 3-yr in situ study to determine the main and interactive effects of nitrogen (N), phosphorus (P), and fungicide (benomyl) addition on EE-GRSP and T-GRSP content in the soil of desert steppe in Northwest China. To further explore the mechanisms influencing GRSP content, the plant community, soil fertility and the AM fungal traits were also identified. Following 3 years of fertilizer and fungicide application, P addition had a negative influence on EE-GRSP content (−12.50%), whereas N addition had no significant effect. Fungicide application reduced EE-GRSP (−18.47%) and T-GRSP content (−18.36%) regardless of N and P addition. By altering the extraradical hyphal length of AM fungi, P and fungicide application decreased the EE-GRSP and T-GRSP content without affecting the vegetation or soil characteristics. Overall, this study provides insights into the dynamics of GRSP in response to soil nutrient enrichment and fungicide application in a resource-limited grassland ecosystem.