Biomass pyrolysis is a promising technology for the production of bio-oil and chemicals. Among these pyrolytic products, furans are important chemicals. In this study, PY-GCXGC-TOFMS was employed to achieve fast pyrolysis of fi-Dglucose and online analysis of the pyrolysis vapors. The effects of pyrolysis temperature on the distribution of main furans are investigated. 3-Hydroxy-gamma-butyrolactone (HBL) and 3-(2H)-furanone (3FO) are observed to be the major C-4 furans which are involved in the evolution of C-4 intermediates in fast pyrolysis. Thus, density functional theory (DFT) calculations and pyrolysis of BC-labeled glucose were combined to reveal possible formation pathways from different intermediates, carbon sources of HBL and 3FO. The results indicated that the production of HBL and that of 3FO are from similar pathways and show a competitive manner in the pyrolysis of glucose. Moreover, HBL and 3FO are confirmed to be mainly derived from C3-C6 of glucose, the possible pathways for the formation of the two furans are also proposed. Mechanism studies here can assist the understanding for the key evolution of C-4 intermediates and contribute to support catalyst design in biomass pyrolysis.