Background and aims: The relationship between transpiration and root distribution under different spatial arrangements of intercropping is poorly understood. The effects of three spatial arrangements in the maize (Zea mays L.) - soybean (Glycine max L.) intercropping on root distribution, transpiration, water use efficiency (WUE) and grain yield were examined. Methods: Two-year field experiments were conducted using three spatial arrangements of 2 rows maize × 4 rows soybean (M2S4), 2 rows maize × 2 rows soybean (M2S2) and 4 rows maize × 2 rows soybean (M4S2), with their respective sole crops (monocrop) for comparison. Results: The grain yield of maize in intercrops was higher than its monocrop and that of soybean in intercrops was lower than its monocrop. Except for M2S2 in 2014, there were yield advantages in intercropping due to improvement in the land use efficiency. Transpiration in maize was higher than in soybean regardless of the spatial arrangements. Transpiration of both maize and soybean was influenced by the spatial arrangements of the intercropping with M4S2 or M2S4 tending to have higher daily transpiration than monocrops and other spatial arrangements. Intercropping enhanced root length density (RLD) in both maize and soybean compared to the corresponding monocrop. RLD was higher and land equivalent ratio (LER) was lower under M2S2 than under other spatial arrangements of intercropping, WUE was higher in M4S2 than in other spatial arrangements. Conclusions: Intercropping was more efficient in using the environmental resources than monocropping. The M4S2 spatial arrangement in the maize-soybean intercropping could be selected because of its sustainability and greater land and water use efficiency.