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Glomalin-related soil protein (GRSP) is an important component of soil organic carbon (SOC), which can promote long-term SOC sequestration. However, GRSP distribution characteristics and its contribution to the SOC pool among different grassland types remain poorly understood. Therefore, six grassland types (alpine meadow, mountain meadow, temperate meadow steppe, temperate steppe, temperate desert steppe, and temperate desert) were chosen to evaluate the contribution of GRSP to the SOC pool and the factors that influence GRSP accumulation in the Irtysh River Basin in China. The results revealed that GRSP (EE-GRSP, T-GRSP) accumulated more in the 0–10 cm soil layer than in the 10–20 cm soil layer (p < 0.05). GRSP content was higher in alpine grasslands (15.69 mg·g−1) than in desert grasslands (5.45 mg·g−1). However, their contribution to the SOC pool exhibited an opposite trend, whereas GRSP-C/SOC even accounted for 11.88% in the desert grasslands. The redundancy analysis (RDA) showed that SOC was the top important positive regulator for GRSP accumulation both in the two layers (explanatory rate > 80%). Besides the SOC factor, the two soil layers had different factors in regulating GRSP accumulation. Changes in GRSP content in the 0–10 cm soil layer were more strongly associated with mean annual temperature (MAT), sand content, soil water content (SWC), and silt content. In contrast, in the 10–20 cm soil layer, GRSP content was more influenced by SWC, electrical conductivity (EC), and pH (p < 0.05). Additionally, the main factor in the GRSP content variation was the interaction between climate and soil in the two soil layers (explanatory rate > 80%). Our findings underscore the critical role of GRSP in facilitating SOC sequestration within desert grasslands and elucidate the primary factors driving GRSP distribution across varying soil depths.