Hydrodeoxygenation (HDO) is usually considered as a promising process for upgrading biomass pyrolysis oil (PO) to bio-fuels. However, cognition of HDO is inhibited by the complexity of the PO and upgraded products. In this study, a PO and its upgraded pyrolysis oil (UPO) samples were analyzed by nuclear magnetic resonance, gas chromatography/mass spectrometry, and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS). ESI FT-ICR MS revealed the most abundant compounds in PO were O-2-O-18 species with double bond equivalent (DBE) values of 0-22. After HDO, oxygen numbers gradually shifted to a range of O-1-O-10, and DBE number also progressively decreased. The major oxygen compounds such as carbonyls, carboxylic acids, ethers, carbohydrates, and alcohols were significantly changed with respect to relative content and molecular composition. Lignin polymers were depolymerized after reduction of the carbonyl and methoxy groups. For HDO, hydrogenation of carbonyls, carbohydrates, and furans occurred under 150 degrees C. Dehydration, hydrodeoxygenation, and dehydration hydrogenation reactions were initiated at 210 degrees C. Decarboxylation and decarbonylation required higher temperatures (>300 degrees C).