Carbon (C) and nitrogen (N) allocation and assimilation are coupled processes, likely influencing C accumulation, N use efficiency and plant productivity in agro-ecosystems. However, dynamics and responses of these processes to management practices in semi-arid agro-ecosystems are poorly understood. A field-based 13CO2 and urea-15N pulse labelling experiment was conducted to track how C and N allocation and assimilation during canola growth from flowering to maturity were affected by short-term (2-year) tillage (T) and no-till (NT) with or without 100 kg urea-N ha−1 (T-0, T-100, NT-0, NT-100) on a Luvisol in an Australian semi-arid region. The T-100 caused greater (P < 0.05) belowground C allocation and higher (P < 0.05) translocation of soil N to shoots and seeds, compared to other treatments. Microbial N uptake was rapid and greatest in the fertilized (cf. non-fertilized) treatments, followed by a rapid release of microbial immobilized N, thus increasing N availability for plant uptake. In contrast, management practices had insignificant impact on soil C and N stocks, aggregate stability, microbial biomass, and 13C retention in aggregate-size fractions. In conclusion, tillage and N fertilization increased belowground C allocation and crop N uptake and yield, possibly via enhancing root–microbial interactions, with minimal impact on soil properties.