A comprehensive understanding of the formation of mineral-associated organic matter (MAOM) is a prerequisite for the sustainable management of soil carbon (C) and the development of effective long-term strategies for C sequestration in soils. Nevertheless, the precise manner by which microbial and mineral properties drive MAOM formation efficiency and its subsequent response to elevated temperature at the regional scale remains unclear. Here, we employed isotopically labelled laboratory incubations (at 15°C and 25°C) with soil samples from a ~3000 km transect across the Tibetan Plateau to elucidate the mechanisms underlying MAOM formation and its temperature response. The results indicated that both mineral protection and microbial properties were critical predictors of MAOM formation across the geographic gradient. The efficiency of MAOM formation was found to increase with the content of iron (Fe) oxides and their reactivity [i.e., the ratio of poorly crystalline Fe oxides to total Fe oxides (Fe_o:Fe_d)] but to decrease with the relative abundance of Gammaproteobacteria and Actinobacteria across the plateau. Moreover, a notable decline in MAOM formation efficiency was observed under elevated temperatures, which was concomitant with a reduction in the content and reactivity of Fe oxides, as well as the microbial assimilation of the labelled substrate. The attenuation of mineral–organic associations was identified as the primary factor contributing to the warming-induced reduction in MAOM formation. These findings highlight the necessity of incorporating organo–mineral associations and microbial properties into Earth System Models to accurately project soil C dynamics under changing climate.