Mechanical activation leads to a variety of defects in MoO3. The behavior of paramagnetic defects in unmilled MoO3 and in MoO3 mechanically activated in a planetary mill for 600 min was investigated by ESR spectroscopy. MoO3 milled for 600 min, and unmilled MoO3 though in a smaller concentration, contain Mo5+ ions; N-2 treatment at elevated temperatures increases the Mo5+ concentration in the samples due to oxygen loss. Both MoO3 samples contain octahedrally coordinated Mo5+ species prior to any treatment which interact with a proton in close proximity. An additional signal is observed in both samples that is characteristic of F centers. An inverse Curie-Weiss behavior between 90 and 300 K indicates the pairing of electrons at lower temperatures. Fivefold coordinated Mo5+ species in distorted, square-pyramidal symmetry are generated in the bulk of unmilled MoO3 during N-2 flushing at 523 and 623 K. MoO3 milled for 600 min contains, besides 6-fold coordinated Mo5+ in C-2 upsilon symmetry, a second Mo5+ species in almost C-4 upsilon symmetry. The Mo-O distances in the equatorial plane are shortened and these Mo5+ centers are assigned to a precursor structure of crystallographic shear planes. Besides B-fold coordinated Mo5+, tetrahedrally coordinated Mo5+ centers are also generated, if milled MoO3 is treated in N-2 at 623 or 673 K which are assigned to the Mo sites on shear defects protruding from the surface. The total intensity of the Mo5+ signals follows the Curie-Weiss law between 90 and 200 K. Above 200 K a deviation from linearity is observed. Hence, a considerable part of the Mo5+ centers does not contribute to the signal intensity at higher temperatures. In situ Raman spectroscopy of mechanically activated MoO3 revealed the formation of some nonstoichiometric MoO3-x after thermal treatment at 673 K in N-2 for 1 h. In addition, diffuse reflectance infrared spectroscopy (DRIFTS) was used to characterize the stability of OH groups on the surface of MoO3 and in the bulk of the mined sample. The OH species located in the bulk of MoO3 was found to be stable even at 673 K under flowing N-2.