Soybean (Glycine max. (L.) Merr.) is a symbiotic nitrogen-fixing crop. In order to increase grain yield, it is important to know how soybean plants respond to nitrogen topdressing for the improvement of nitrogen utilisation. We used two soybean cultivars with different grain yield potentials and applied 13 nitrogen topdressing treatments to determine optimal topdressing time and nitrogen metabolism. Nitrogen treatments included a base fertiliser and single topdressings at different times, in 10-day intervals from 10 to 120 days after emergence (DAE). Among the nitrogen treatments, the optimal times for topdressing were at 40 DAE or 90 DAE to increase grain yield, and both soybean cultivars also had higher nitrate reductase (NR) and glutamine synthetase (GS) activities with topdressing at these times. Higher expression of the NR2 gene was associated with upregulated NR activity in leaves of both cultivars at the early-mature stage. With topdressing at 90 DAE, higher GS1 expression and GS activity were found in the leaves of the higher yielding cultivar at the full-seed stage and the early-mature stage. With topdressing at 90 DAE, the higher yielding cultivar had a higher nitrate metabolism capacity at the late reproductive stages than the lower (common) yielding cultivar.