A layered structure of orthorhombic MoO3 (α-MoO3) was investigated as a new electrode material for calcium-ion batteries in a Ca-based organic electrolyte. Ground α-MoO3 with a low reactivity carbon additive demonstrated a relatively high capacity of ~120 mAh/g per one-electron reaction even though a large irreversible capacity was obtained because of electrolyte decomposition during the 1st discharge (Ca2+ insertion). In addition, the X-ray diffraction (XRD) patterns after the 1st discharge and charge showed decreased intensities of the 0k0 reflections with increased peak widths in comparison with the XRD patterns of an as-prepared pellet, which suggests that structural damage occurs in the layered structures. However, the XRD patterns, energy-dispersive X-ray (EDX) spectra and X-ray photoelectron spectra (XPS) of the discharged and discharged–charged electrodes indicated that Ca2+ ions are inserted into and extracted from the crystal structures via a two-phase reaction.