In strongly correlated materials, change of the local lattice configuration is expected to tune or even generate new properties otherwise in the ideal bulk materials. For highly spin-polarized materials, the spin-dependent transport is sensitive to the local magnetic structure. Here, the artificial grain boundaries (GBs) with different tilt angles are produced in Fe3O4 films using SrTiO3 bicrystal substrates. The saturation magnetization of Fe3O4 bicrystal films is enhanced. The detailed atomic structural results combining with density functional theory calculations reveal that the elongated FeA-O bond length at GBs resulting in the reduction of charge transfer reduces the FeA magnetic moments, which enhances the total magnetic moments of Fe3O4. The in-plane rotation of the Fe3O4 lattice on bicrystal substrates alters the magnetization processes. Especially, the Fe3O4 bicrystal film with a tilt angle of 22.6° shows strong in-plane magnetic anisotropy due to the zigzag GBs. The altered magnetic anisotropy of Fe3O4 bicrystal films enhances the anisotropic magnetoresistance. The findings reveal the mechanism of GBs on the magnetic and transport properties and manifest that the strategy of utilizing GBs can tune the physical properties in highly spin-polarized materials.