Spatial heterogeneity is ubiquitous in soil systems. The spatially heterogeneous distribution of labile organic carbon substrates in soil can alter microbial accessibility to carbon sources and their spatial matching relationship with nutrients, thereby regulating soil CO2 emission. However, whether the inhibitory effect of substrate spatial heterogeneity on soil CO2 emission depends on inter-layer material connectivity remains unclear. This study aimed to reveal how substrate spatial heterogeneity and inter-layer material migration regulate soil CO2 emission.Using a typical subtropical red soil as the research object, a laboratory incubation experiment was conducted in which soils were arranged in horizontal layers. Under the condition of identical total exogenous carbon input, glucose was added to the horizontally layered soil columns in different ways to establish four treatments: high heterogeneity, low heterogeneity, homogeneity, and a no-carbon-added control. Two migration modes—inter-layer isolation and diffusion—were set up to monitor the dynamics of soil CO2 emission. Contents of dissolved organic carbon (DOC), microbial biomass carbon (MBC), ammonium nitrogen (NH4+-N), and nitrate nitrogen (NO3--N) in each soil layer were measured to explore the effects of substrate spatial heterogeneity on soil CO2 emission and on the spatial distribution of labile carbon and nitrogen components across layers.The results showed that, compared with the no-carbon-added control, all carbon-amended treatments increased soil CO2 emission. However, under equal carbon input, the heterogeneous treatments exhibited lower cumulative CO2 emission than the homogeneous treatment, and the magnitude of decrease increased with the degree of heterogeneity. This heterogeneity-induced inhibitory effect was modulated by inter-layer material migration: differences in CO2 emission among the heterogeneous treatments were more pronounced under the isolation condition, whereas diffusion-mediated inter-layer material migration partially weakened the suppression of carbon mineralization by heterogeneity. Nutrient distribution patterns indicated that DOC and MBC contents were significantly higher in substrate-amended layers than in unamended layers, whereas the reverse was true for NH4+-N. The coefficients of variation of DOC, MBC, and NH4+-N across soil layers were generally higher in the heterogeneous treatments than in the homogeneous treatment, and were slightly higher under isolation than under diffusion.This study demonstrates that the spatial differentiation of labile carbon, inorganic nitrogen, and microorganisms across soil layers caused by the spatial heterogeneity of labile organic carbon substrates reduces total soil CO2 emission, and that inter-layer material migration can partially alleviate this inhibitory effect.