Soybean has the ability to live in symbiosis with microorganisms and take up nitrogen from the atmosphere, fertiliser and soil reserves. The amount of nitrogen taken up from these sources depends on many biotic and abiotic factors, e.g., the rhizobium species, cultivar, as well as weather and agricultural conditions. A field experiment was conducted in eastern Poland (central Europe) in two successive growing seasons to examine the uptake of nitrogen from the atmosphere (NDFA—% nitrogen derived from the atmosphere), fertiliser (NDFF—% of nitrogen derived from fertiliser) and soil reserves (NDFS—% of nitrogen derived from the soil) for three non-GMO (non genetically modified organism) soybean cultivars: Abelina, SG Anser and Merlin. Pre-plant fertilisation of plants with nitrogen excess with the 15N isotope and the isotope dilution method were applied. Soil reserves and the atmosphere were major nitrogen sources for soybean. Soybean roots contained the most atmosphere-derived nitrogen (45.85%), the amount being lower for soil reserves (41.43%) and the lowest for fertiliser (12.72%). Harvest residues and seeds contained the most soil reserve-derived nitrogen, the amount being lower for the atmospheric nitrogen and the lowest for fertiliser-derived nitrogen. The amount of nitrogen derived from different sources in the whole soybean mass significantly depended on cultivars and years’ percentage values being affected by study years only. Less atmospheric nitrogen was accumulated in cv. Abelina roots (2.15 kg N·ha−1) compared with cv. SG Anser (3.07 kg N·ha−1) or cv. Merlin (2.89 kg N·ha−1). More atmospheric nitrogen was recorded in the post-harvest residues and seeds of cv. Abelina and SG Anser than Merlin. The content of soil reserve-derived nitrogen taken up by the whole soybean plants averaged 61.29 kg N∙kg−1, the amounts being 50.95 and 11.38 kgN∙kg−1 for nitrogen taken up from the atmosphere and fertiliser, respectively. Soybean grown in the study year with more favourable thermal and precipitation conditions (2017) took up more nitrogen from all the sources compared with the year 2018.