The effect of antisite disorder in the layered sodium iron antimonate Na4FeSbO6 was examined both experimentally and theoretically. The magnetic susceptibility and specific heat measurements show that Na4FeSbO6 does not undergo a long-range antiferromagnetic order, unlike its structural analog Li4FeSbO6. The electron spin resonance yields the complicated picture of coexistence of two magnetic subsystems corresponding to two different Fe cation positions in the lattice (regular and antisite) and driving the magnetic properties of the Na4FeSbO6. This conclusion found perfect confirmation from both the Mössbauer and X-ray absorption data which show the presence of two kinds of Fe3+ ions being in high-spin Fe3+ (S = 5/2) and low-spin Fe3+ (S = 1/2) states. These findings arise from the antisite disorder between the Fe3+ and Sb5+ ions in the (NaFeSbO6)3− layers of Na4FeSbO6. Our density functional calculations show that the Fe3+ ions located at the Sb5+ sites exist as low-spin Fe3+ ions, and that the spins of each Fe3+ (S = 5/2)–Fe3+ (S = 1/2)–Fe3+ (S = 5/2) trimer generated by the antisite disorder has a ferrimagnetic arrangement Fe3+↑–Fe3+↓–Fe3+↑, which enhances the magnetization of Na4FeSbO6 and leads to an apparently positive Curie–Weiss temperature.