The traditional view holds that the global distribution of biological nitrogen fixation (BNF) in terrestrial ecosystems is primarily driven by climate, associating certain warm, wet low-latitude regions with higher BNF rates. However, this view fails to explain the low free-living BNF rates observed in these regions. Here, we conducted two field experiments and a global synthesis to assess the regulatory patterns of free-living BNF at both regional and global scales. The field experiments showed that the regional distributions of litter free-living BNF did not necessarily peak at warm and wet sites, due to co-regulation by climate and substrate stoichiometry (especially C : [N : P]). Subsequently, the global synthesis demonstrated that these phenomena and co-regulatory patterns persisted in free-living BNF in litter and other substrates (e.g., soil) at global scale. Our findings highlight the important role of substrate stoichiometry in regulating the spatial distribution of free-living BNF.