Land-use change alters soil carbon (C) storage in terrestrial ecosystem through affecting soil respirations (Rs). However, the long-term effects of land-use change on Rs are poorly understood. We undertook a field experiment to examine the effects of long-term land-use change on Rs. The land-use types included grazed grassland (GGL), enclosed grassland (EGL), continuous cropland (CCL), rotation cropland (RCL) and abandoned cropland (ACL) in an alpine agro-pastoral ecotone in Qinghai-Tibetan Plateau in China. Our results showed that the accumulative Rs was shown a decreasing order of EGL > CCL > RCL > GGL > ACL, with the highest value of 12.2 t CO2 ha−1 and the lowest value of 5.3 t CO2 ha−1. Soil bulk density, moisture, pH and C/N ratio were the key factors regulating Rs with land-use change in Tianzhu alpine agro-pastoral ecotone. Soil pH and C/N ratio regulated Rs indirectly by affecting bacterial diversity, while soil moisture had indirect impacts on Rs mainly by regulating microbial biomass. The net effects of soil bulk density on Rs was mainly through the direct pathway. Considering the soil C stocks showed an increasing order of ACL < RCL < CCL < GGL < EGL, even though ACL had the lowest Rs, grasslands probably the favorable land-use type in alpine agro-pastoral ecotone. Our findings highlight the importance of the land-use change on Rs, which may need to be incorporated into regional and global models for better predicting C-climate feedbacks.