We examined whether glomalin-related soil protein (GRSP), produced by arbuscular mycorrhizal fungi (AMF), and with role in soil quality, structuring, and carbon storage, varies in relation to substrate and plant community in the campo rupestre ecosystem. Soil samples were collected for physical-chemical and GRSP content analysis, in 40 plots, where the vegetation was also surveyed. GRSP content was compared between substrate types, and its relationship was assessed against soil attributes, vegetation dissimilarity, and abundance of non-mycorrhizal species and species with low AMF dependency. GRSP content varied greatly among the sites (total fraction ranging from 4.66 to 135.46 mg g−1), differed significantly between ferruginous and quartzitic substrates, and was correlated with soil organic carbon (SOC), soil nutrients, soil texture, and vegetation dissimilarity. Total glomalin contributed about 20% to the SOC. GRSP content declined with increasing cover of species that are both sand-binding and have AMF associations, and non-mycorrhizal species. The study shows how AMF, through GRSP, is associated to the ecosystem heterogeneity, suggesting the GRSP as an important soil factor to be considered in management and conservation issues in campo rupestre and similar ecosystems. GRSP content was shown to contribute to carbon storage and was linked to soil chemical and physical properties, and affected by variation in cover of non- or less-AMF-dependent plant species, perhaps providing a useful indicator in the heterogeneous campo rupestre system.