Vast subtropical gyres are important areas for the exchange of carbon between atmosphere and ocean in spite of low nutrient concentrations, and supposedly for the influx of reactive nitrogen to the ocean by dinitrogen fixation. To identify sources and transformation processes in the nitrogen cycle of the southern Indian Ocean subtropical gyre, we investigated concentrations of water column nutrients and stable isotope composition of nitrate of samples from two expeditions in 2016 (MSM 59) and 2017 (SO 259) in the subtropical gyre between ~ 30 °S and the equator. Low nitrate and phosphate concentrations mark the thick mixed layer of the oligotrophic gyre with values of < 5.9 µM NO<sub>3</sub><sup>−</sup> and < 0.5 µM PO<sub>4</sub><sup>3−</sup> (< 310 m; σ < 26.4 kg/m³). Increased nutrient concentrations towards the equator represent the northern end of the gyre, characterized by typical strong horizontal gradients of the outcropping nutriclines. Measurements of stable isotopes of nitrate (δ<sup>15</sup>N and δ<sup>18</sup>O) indicate isotopic maxima of δ<sup>15</sup>N (> 7 ‰) and δ<sup>18</sup>O (> 4 ‰) centred at 400–500 m, representing the preformed nitrate exported from the Southern Ocean with mode water and induced by partial N-assimilation there. Additionally, a residue of nitrate affected by denitrification in the Arabian Sea is imported into the sub-thermocline of the gyre, indicated by a strong N deficit (N* < −1 µM) within the northern study area, accompanied by elevated isotopic ratios of nitrate (δ<sup>15</sup>N > 7 ‰; δ<sup>18</sup>O > 3 ‰). The subtropical South Indian Ocean is thus supplied by nitrate from lateral influx of water masses that have similar isotopic character, but antagonistic origin (preformed versus regenerated). A significant contribution of N<sub>2</sub>-fixation within the Indian Ocean subtropical gyre (17° S–25° S) is promoted by low nitrate to phosphate ratios in the surface layer, where approximately one-third of the nitrate in the upper ocean is derived by newly fixed N.