Willow (Salix spp.) is currently being researched as a source of biomass energy in Canada. However, it is not certain whether afforestation with willow plantations will enhance or diminish soil C storage and nutrient availability. Trees are known to have pronounced effects on biologically mediated nutrient cycling processes which can increase nutrient availability, but willows are known to be nutrient demanding. In this paper, the net effect of plantation establishment is examined at nine sites across the prairie and southern Ontario regions of Canada. Carbon, N, P, K, Ca and Mg levels in soils and harvestable biomass were compared between willow plantations and paired reference sites at the end of the first three-year rotation. Soils were depleted in total C (−2.22 mg·g −1 , p < 0.05), inorganic N (−3.12 μg·N·g −1 , p < 0.10), exchangeable K (−0.11 cmol c ·kg −1 , p < 0.10) and leachable P (−0.03 mg·g −1 , p < 0.10). Exchangeable Ca was found to be consistently depleted only at the 20-40 depth. Depletion of soil K was more heavily influenced by disturbance, whereas soil N was directly affected by willow N uptake. Sites with greater growth and biocycling stabilized soil P concentrations.