Little is known how the deep root systems of perennial crops contribute to deeper and better resource use when intercropped with annuals in arable fields. Therefore, we aimed at measuring the capacity of deep-rooted perennials, alfalfa (Medicago sativa L.) and curly dock (Rumex crispus L.) to access nutrients located under neighboring annuals at 1.0 and 2.5 m of soil depth. Alfalfa and curly dock were able to access the tracer sources (¹⁵N and Cs) placed at a distance farther than 0.4 m under the annual crop strips. As a result, the reliance on deeper soil layers for nutrient uptake by the crops grown at the borders became greater compared with the crops grown in the mid-strips. Combination of an annual cereal (winter rye) and a perennial legume (alfalfa) having contrasting root systems exhibited higher resource complementarity compared with intercroppings having more similar root systems or in the absence of legumes. Our results demonstrated that the deep-rooted perennials, when intercropped with annuals, can induce vertical niche complementarity, especially at deeper soil layers. This was assumed to be due to the differences in rooting depth and deep root activity the crop components, however, the magnitude of the effects depended on choice of crop combinations, and on types of tracers used in the study.