MoO3 nanofibers have been synthesized by hydrothermal reaction from acidified ammonium heptamolybdate tetrahydrate precursor. Characterization included X-ray diffraction (XRD), scanning electron microscopy (SEM), SQUID magnetometry and Raman scattering (RS) spectroscopy. Structural analysis shows that MoO3 nanofibers 50–80 nm in diameter and a several micrometers in length are grown in the orthorhombic system (Pbnm S.G.). The composition MoO2.9975 was determined by Rietveld refinement and magnetic susceptibility measurements. The electrochemical performance of Li//MoO3 cells with nanofibers deliver a better discharge capacity after 40 cycles than MoO3 bulk. Raman spectroscopy studies revealed the structural modulation upon Li intercalation in MoO3 nanofibers. The possible mechanism of intercalation is discussed.