Natural exchanges of carbon (C) between the atmosphere, the oceans, and terrestrial ecosystems are currently being modified through human activities as a result of fossil fuel burning and the conversion of tropical forests to agricultural land. These activities have led to a steady increase of atmospheric carbon dioxide (CO 2) over the last two Centuries. The goal of this study was to determine the potential of temperate agroforestry systems to sequester C in soil. Therefore, changes in the soil organic C (SOC) and nitrogen (N) pools were quantified and the δ 13C and δ 15N stable isotope technique was applied to assess soil C and N dynamics in a 13-year old hybrid poplar alley cropping system in Southern Canada. Results from this study showed that after 13 years of alley cropping the SOC and N pools did not differ significantly ( p = 0.01) with distance from the tree row, although a trend of a larger SOC and N pool near the tree row could be observed. Soil organic C after 13 years of alley cropping, was 19 mg C g −1 compared to 11 mg C g −1 upon initiation of agroforestry. Soil organic C and N were not evenly distributed throughout the plow layer. The largest C and N pool occurred in the top 20 cm, which is due to the accumulation of organic material in the upper horizons as a result of no-till cultivation. The entire soil, to a 40 cm depth, showed a δ 13C shift to that of C 3 residue. This shift reflects the greater input of residues from C 3 plants such as that derived from beans, wheat, and hybrid poplar leaf litterfall. The proportion of C derived from a C 3 source ranged from 64 to 69% to a 40 cm depth. The soil δ 15N signature of this study is similar to that of mineral soil, and reflect values characteristic of N mineralization processes. However, the entire soil shows a positive shift in δ 15N as a result of historical additions of manure and current use of mineral fertilizers, and ongoing processes of denitrification and nitrate leaching, which leads to an enrichment of the soil.