Uncertainty remains concerning the magnitude of symbiotic N fixation by the soybean crop [Glycine max (L.) Merr.] when grown under varied N management systems. For deriving further enlightenment on the issue, 15N‐enriched fertilizer was applied on two soybean isolines with objectives of measuring the fraction of N derived from symbiotic fixation, residual soil N and applied fertilizer N. Plant samples were collected at full bloom, beginning‐seed, and full‐maturity stages in a field experiment conducted at the University of Nebraska Field Lab on Sharpsburg soil (Typic Argiudoll) of 3.3% organic matter content. Nitrogen fertilizer was applied at rates of 45, 89, and 134 kg/ha at planting or at full bloom. Nitrogen fertilization had no significant influence on yield or N and oil concentration of the nodulating isoline seed but increased these parameters of the non‐nodulating isoline, with delay in N fertilization being distinctly advantageous. Fertilizer utilization percentage by both isolines at later growth stages increased as fertilizer rate increased, contrary to N utilization patterns of cereal crops. “A ” value increases with increased N fertilizer rates suggested a soil N priming and/or root extension effect. Less plant N came from the soil with delayed N fertilizer application. Fertilizer applied at planting at rates above 45 kg N/ha reduced the symbiotically fixed N fraction, whereas delaying fertilizer application had no such influence at any rate of N employed.