In this work, the role of Gd3+ ion content on the structure and magnetic properties of Fe3-xGdxO4 (x = 0.00, 0.06, 0.12, 0.18, 0.24, 0.30) nanoparticles (NPs) are comprehensively evaluated by x-ray diffraction (XRD), Field Emission Scanning Electron Microscopy (FESEM), Mossbauer, and M-H and zero-field cooling curves. Analysis of experimental results reveals that the diameter of the substituted NPs ranged from 12.5 nm to 16.8 nm, which is smaller than that of pristine Fe3O4 NPs (18.7 nm). The substitution of Gd3+ ions in Fe3O4 crystalline occurs mainly at the A-site (x = 0.06, 0.18) or at the B-site (x = 0.12, 0.24). This led to an increase in lattice parameter (8.359–8.395 Å) and density (5.41–6.04 g cm−3) as well as the change in the inversion parameter. As a result, the substituted NPs' diameters (12.5 nm—16.8 nm) are smaller than that of pristine Fe3O4 NPs (18.7 nm). In particular, the highest experimental saturation magnetization (73.5 emu g−1 at 300 K) because of the largest size, smallest value of the inversion parameter, theoretical and experimental value of the total magnetic moment, and the main doping at the B-site. This phenomenon and all data on saturation magnetization governed by Bloch's T3/2 law confirm the highest effect of Gd3+ ions on the magnetic properties of Fe2.82Gd0.18O4 NPs. All results indicate that the effect of Gd content on the structure and magnetic properties of Fe3-xGdxO4 NPs depends strongly on the position of substituting Gd ions and corresponding changes in the inversion parameter.