Soil aggregates are the ‘building blocks’ of soil structure. Aggregation dominantly influences the physico-chemical and biological properties of soil. Farm management practices can alter soil aggregation, which can potentially influence carbon dioxide (CO2) fluxes. We examined the effect of soil aggregate size on CO2 fluxes in a Vertisol following 47 years of contrasting management regimes, namely no-till (NT) or conventional till (CT), stubble retention (SR) or burning (SB), and without nitrogen (N) application (0 N) or with 90 kg N ha−1 year−1 (90 N). Aggregate size significantly affected CO2 fluxes over a 21-day incubation period, with the smallest aggregate size of <2 mm having the highest cumulative CO2 flux, followed by the 2–4 mm then 4–8 mm aggregates. The observed differences were mainly caused by total organic carbon contents, as well as substrate accessibility. Over all aggregate sizes, the SR treatments had higher CO2 fluxes compared to SB treatments due to additional substrate availability for microbial respiration. Cumulative CO2 flux was higher for the N fertilised treatments than the unfertilised treatments, likely as a result of increased soil organic matter content and the reduced C:N ratio. Fluxes of CO2 from small and intermediate aggregates were lower for NT compared to CT when 90 N fertiliser was applied, but not significantly different without N fertiliser application. This study identified the underlying controlling factors of CO2 flux from different aggregate sizes in a Vertisol under long term contrasting management regimes.