Relay strip intercropping of soybean has been widely developed in the southwest of China to secure China's soybean production. However, due to the shading from maize, soybean plants are thin and have a poor root system. Uniconazole is a plant-growth retardant that could enhance root vigor; increase root length, root volume, and root dry weight; and affect nitrogen (N) metabolism. To understand the effects of uniconazole on the root growth and N-transfer metabolism of soybean seedlings under relay strip intercropping, the changes in some morphological characteristics of root, dry-matter weight, root vigor, nitrate (NO3 −)-N, ammonium (NH4 +)-N, and amino acid of xylem sap after seed treatment with uniconazole powder (0, 2, 4, and 8 mg kg−1 seed) were investigated. Main root length, total lateral root lengths, first lateral root numbers, root nodule numbers, root vigor together with bleeding sap, bleeding sap–top ratio, root dry weight, and root/shoot ratio were increased, indicating uniconazole improved soybean root system in relay strip intercropping. Uniconazole powder treatment could increase NO3 −-N, NH4 +-N, and total amino acid of xylem sap, to increase the potential of leaf and root N reduction and assimilation, and increase of leaf and root N contents. Thus, results suggested that uniconazole treatment can improve root growth and N transfer mechanism of soybean to support its further growth.