Tree root pruning in agroforestry could reduce water competition and increase the crop yield. The aim of this study was to evaluate the impact of tree root pruning on the yield of winter cereals in a mature Mediterranean alley cropping system considering crop phenology and the position in the alley. An experiment was conducted in a walnut alley cropping. Two modalities were established: root pruning (RP+) and no root pruning (RP-). In each one four genotypes of winter cereals were sown. Microclimate, soil matric potential (SMP), crop phenology and yield components were measured. The SMP presented higher values in RP+, especially in the central part of the alley. The impact on crop phenology of the root pruning and the position in the alley varies according to the genotype. The barley yield was statistically higher in RP+, whereas wheat yield did not show significant differences between modalities.