The influence of mechanical activation in a planetary mill upon the nature and concentration of defects in MoO3 powders was investigated by means of diffuse reflectance spectroscopy in the UV/vis regime (DR-UV-vis) and by electron spin resonance (ESR). Defects located at the crystallite surfaces were characterized by infrared spectroscopy of adsorbed CO as a probe molecule. For this purpose, MoO3 was ball-milled in a planetary mill during 600 min. In the DR-UV-vis spectra, the valence-to-conduction band transition exhibits a considerable blue shift with decreasing particle size. Furthermore, excitonic absorptions observed in these spectra are also drastically affected by the smaller particle size and probably by the altered crystallite surfaces. An increasing intensity of the polaron bands was observed. In addition, a linear dependence was obtained between the position of the band attributed to polaron conductance and the logarithm of the carrier concentration per Mo atom. Both the increasing intensity and the shift of the polaron band revealed that a substoichiometric MoO3-x was formed upon mechanical treatment. ESR spectroscopy showed that MoO3 milled for 600 min, and unmilled MoO3 although in much smaller concentration, contained Mo5+ centers. The main part of these Mo5+ ions had C-2v or C-4v symmetry. Both samples also contained Mo5+ centers interacting with protons in close vicinity. Adsorption of O-2 did not lead to paramagnetic broadening; hence these Mo5+ centers are located within the bulk MoO3. In addition, a signal in the ESR spectra of both samples is assignable to free electrons at the crystallite surfaces as revealed by paramagnetic broadening upon O-2 adsorption. One Mo5+ defect species, however, was only detected in milled MoO3 and attributed to the precursor structure of shear defects, thus corroborating the reported XRD and HRTEM results. The high surface sensitivity of the IR technique using adsorbed probe molecules revealed the formation of coordinatively unsaturated (cus) Mo4+ surface states in MoO3 samples which were mechanically activated.