Twin defects, prevalent in face centred cubic stacked materials, are observed across a wide range of natural and synthetic specimens. Such defects are essential to the mechanical behaviour of materials e.g. shape memory alloys and mediate stress and strain in both functional and mechanical materials. Fe 3 O 4 is a prototype material for spinel metal oxide structure systems, particularly the ferrite spinels. Recently magnetite has attarcted a lot of attention due to its 100% spin polarisation at the Fermi level, hence large intestet for spintronic applications. Antiphase domain boundaries are the most studied defects in magnetite and theior correlatin to magnetic properties is reported extensively [1,2]. However twins defects magnetite has not been studied on atomic level, and in particular their effect on spin polarisation and overall magnetic properties is not known. In this we have observed twin defects in Fe 3 O 4 (111) thin films grown on Ytria stablized ZrO 2 , determined their atomic structure, and modelled their electronic properties by using Density Functional Theory (DFT).The twin boundary is on (111) growth planes and it is formed by the breaking of symmetry of a Fe AFe B -Fe A layer. Aberration-corrected STEM-HAADF imaging shows that the boundary is nonstoichiometric with a missing Fe B plane. Electron energy loss spectroscopy shows changes in Fe and O core edges as well as depletion of the Fe at the boundary, which compliments the HAADF results (Fig.1). The DFT calculation of this non-stoichiometric boundary structure was modelled by introducing electron holes as a charge-compensation mechanism to realise the ionic nature of Fe 3 O 4 , Fig. 2. The electronic calculations show that majority band gap is significantly reduced with a presence of the interface states. Atomic bond counting from the DFT-optimised geometrical coordinates shows no presence of high-angle Fe-O-Fe bonds hence the absence of AFM superexchange interactions at this boundary in comparison to antiphase domain boundaries in ferrite spinels. The ferromagnetic (FM) coupling between the twin grains was also confirmed by the DFT calculation which found that AFM coupling is less energetically favourable compared to FM coupling. This work clearly shows that the (111) oriented non-stoichiometric boundaries are energetically stable and their effect is more subtle in comparison to the antiphase domain boundaries [3].