IntroductionSurplus use of chemical nitrogen (N) fertilizers to increase agricultural Q9 production causes severe problems to the agricultural ecosystem and environment. This is contrary to N use efficiency and sustainable agricultural production.MethodsHence, this study was designed to investigate the effect of maizesoybean intercropping on N uptake, N yield, N utilization use efficiency, and the associated nitrogen assimilatory enzymes of maize crops under different N fertilization for two consecutive years 2021-2022.ResultsThe findings of the study showed that intercropping at the optimal N rate (N1) (250 kg N ha-1) increased significantly maize grain yield by 30 and 34%, residue yield by 30 and 37%, and 100-grain weight by 33 and 39% in the year 2021 and 2022, respectively. As compared with mono-cropping, at this optimal N rate, the respective increase (of maize’s crop N yield indices) for 2021 and 2022 were 53 and 64% for grain N yield, and 53 and 68% for residue N yield. Moreover, intercropping at N1 resulted in higher grain N content by 28 and 31%, residue N content by 18 and 22%, and total N uptake by 65 and 75% in 2021 and 2022, respectively. The values for the land equivalent ratio for nitrogen yield (LERN) were greater than 1 in intercropping, indicating better utilization of N under the intercropping over mono-cropping. Similarly, intercropping increased the N assimilatory enzymes of maize crops such as nitrate reductase (NR) activity by 19 and 25%, nitrite reductase (NiR) activity by 20 and 23%, and glutamate synthase activity (GOGAT) by 23 and 27% in 2021 and 2022, respectively. Consequently, such increases resulted in improved nitrogen use efficiency indices such as N use efficiency (NUE), partial factor nitrogen use efficiency (PFNUE), nitrogen uptake efficiency (NUpE), and nitrogen agronomic efficiency (NAE) under intercropping than mono-cropping. ConclusionThus, this suggests that maize-soybean intercropping under optimal N fertilization can improve the nitrogen status and nitrogen use efficiency of maize crops by regulating the nitrogen assimilatory enzymes, thereby enhancing its growth and yield. Therefore, prioritizing intercropping over an intensive mono-cropping system could be a better option for sustainable agricultural production.