Stable water isotope tracing is an effective tool for quantifying plant water sources; however, interspecific competition and root stratification mechanisms in intercropping systems remain insufficiently validated through isotopic methods. Thus, to address this gap, a field study in semi-arid northwest China (Baiyin, Gansu; 209 mm annual precipitation) was carried on wheat-maize intercropping system encompassing treatments of monoculture wheat, monoculture maize, and intercropping. Water distribution mechanisms were evaluated using hydrogen-oxygen isotopes (delta H-2/delta O-1(8)) combined with the MixSIAR model. The results demonstrated that intercropping provided synergistic benefits through vertical root stratification, whereby wheat primarily accessed shallow soil water, while maize relied on deep water layers. Net area yields significantly increased under intercropping for both wheat (7877 kg ha(-1), +17 %) and maize (27,959 kgha(-1), +58 %) compared to monoculture. Net water use efficiency of intercropped maize increased by 45 %. Border row wheat exhibited a more balanced water uptake across soil layers (shallow: 33 %, middle: 36 %, deep: 31 %), enhancing biomass accumulation and yield components. Middle-row maize showed 75 % higher flowering-stage biomass than border rows, attributed to preferential water use from middle soil layer (28 %), which reduced interspecific competition. Post wheat harvest, deep soil water storage in the maize strip increased by 8 %. A 30 cm row spacing effectively minimized border row competition (competition intensity=0.45). We recommend applying shallow-layer irrigation at the wheat jointing and flowering stages (fulfilling 80 % of water demand) and deep-layer irrigation during the maize milk stage (increasing soil water storage by 8 %), along with maintaining a 30 cm row spacing. This integrated strategy significantly increased yields (>17 % for wheat and >50 % for maize) and provides a quantitative basis for designing water-efficient intercropping systems in arid regions.