In situ prepared Fe3O4(100) thin films were studied by means of scanning tunneling microscopy (STM) and spin-polarized photoelectron spectroscopy (SP-PES). The atomically resolved (2×2)R45°wavelike surface atomic structure observed by STM is explained based on density functional theory (DFT) and ab initio atomistic thermodynamics calculations as a laterally distorted surface layer containing octahedral iron and oxygen, referred to as a modified B layer. The work-function value of the Fe3O4(100) surface extracted from the cutoff of the photoelectron spectra is in good agreement with that predicted from DFT. On the Fe3O4(100) surface both the SP-PES measurements and the DFT results show a strong reduction of the spin polarization at the Fermi level (EF) compared to the bulk density of states. The nature of the states in the majority band gap of the Fe3O4 surface layer is analyzed.