[Purpose] The maize soybean relay strip intercropping systems (MS) can improve nitrogen (N) uptake. N uptake mechanisms require further study to better understand how different interspecific row spacing affect root physiological activity and to determine crop’s N utilization. [Methods] A two-year field experiment was conducted with two N application levels referred to as no N application (NN) and conventional N application (CN) were paired with different interspecific distance, including 30, 45, 60, 75 cm (MS30/45/60/75), and 100 cm of monoculture maize and soybean (MM/SS100). [Results] Compared to MM100, MS30 increased root activity and respiration intensity by 35.8%, 36.6%, antioxidant enzyme activity, and the relative expression levels of genes encoding antioxidant enzymes markedly improved. The NO3−-N and NH4+-N uptake increased 83.5% and 39.4%, the N uptake rate improved and the maximum achievable total N uptake (Nmax) increased 49.4% under NN, 20.7% under CN. Relay intercropping soybean in MS60 increased root activity by 63.5%, enhanced antioxidant properties, and root N uptake, shoot N accumulation notably increased than SS100. The N uptake rate of MS60 exceeded SS100 at 70 days after sowing (DAS), and the Nmax increased 39.8% than SS100. The correlation analysis indicated aboveground N uptake were positively correlated with root physiological activity. [Conclusions] Therefore, optimizing crop configuration achieved N accumulation increase, N uptake rate accelerate and better N balance by promoting root physiological activity in MS. The suitable interspecific row spacing (45–60 cm) can coordinate the advantages of N uptake to achieve optimal N use and land productivity in relay intercropping system.