In this work we analyze the role of the SiO2 layer in the functionality of Fe3O4/SiO2/Si heterostructures, which have been proved to present a strong potential for spin-based applications. Nevertheless, a complete control of the interfaces properties is fundamental for application. In this work, high quality heterostructures are fabricated avoiding chemical exchange and achieving good quality interfaces. The chemical interaction between the Fe3O4 and SiO2 layers during the heterostructures manufacture is deeply analyzed and its role on the transport, magnetic and magneto transport response is revealed. It is proven that during Fe3O4 deposition a competitive interplay happens between the catalytic action of Fe atoms, the transport of dissociated oxygen through SiO2 and the stabilization of Fe3O4. A defective silicon oxide layer is found to grow on top of the native SiO2 enabling the formation of single phase Fe3O4 layer. Such a defective layer and the granular character of the Fe3O4 determine the magnetic and transport response of the heterostructures. The present results prove that the defects in the SiO2 layer induce the switching of the MR sign, so that anomalous positive MR at RT exceeding 17% at 80 kOe is obtained in heterostructures with 19 nm thick magnetite layer, while conventional negative MR response is obtained for thicker films.