Coumarin is one of the basic structures of naturally oxygen heterocyclic compound, which was investigated in this paper for its gas-phase fragmentation behaviors using electrospray quadrupole extractive orbitrap mass spectrometry in the positive mode.The possible fragmentation pathways were proposed based on electrospray ionization (ESI)-mass spectrometry (MS)/MS data and theory calculation. The elimination of two CO and CO 2 was observed for protonated coumarin, which was followed by the formation of a stabilized seven-, six-, and five-membered ring carbocation by loss of C2H2. The possible protonation sites occurred at Oxygen 11 atom of coumarin were the main fragmentation pathways. The relative abundance of characteristic fragment ions and the energy-resolved breakdown curves were used to confirm the cleavage mechanism of protonated coumarin. The methodology and results of present work would contribute to the chemical structure identification of other coumarins. K E Y W O R D S breakdown curves, fragmentation pathways, coumarin, DFT calculations MASS SPECTROMETRY 6 of 9 SUN ET AL. Journal of MASS SPECTROMETRY time, it should not emerge that ion D5 (m/z 91) transform into A (m/z 147), because two CO are respectively lost in path (b). After 55 eV, the relative abundance of product ions C (m/z 103) and D (m/z 91) may be decided by the above-mentioned cause. According to the maximal ΔG' of losing C 2 H 2 in Figure 3 (67.3 kcal/mol), which was lower than that of losing first CO (69.7 Kcal/mol) in Figure 1, the relative abundance of ion F (m/z 65) should be higher than B (m/z 119) and D (m/z 91). The results of relative abundance of ion F was lower than D before 140 eV, and higher after 140 eV in Figure 6. According to the maximal ΔG' in Figure 3, the maximal ΔG' of ion D (m/z 91) transformed into F (m/z 65) (67.3 kcal/mol, from ion D to TD 1 , black line) was higher than D (m/z 91) to D5 (m/z 91) (61.3 kcal/mol, from ion D to TD 4 , red line). So the process ion D to D5 was the favorite path and the result consistent with the curve of ions D (m/z 91) and F (m/z 65) when the NEC was lower than 140 eV. For the same reason, the red line part in Figure 3 (ion D transformed into D5) was a reversible reaction, on which the ions D (m/z 91) and D5 (m/z 91) could be transformed into each other. On the other hand, the black line part in Figure 3 would be an irreversible process, with losing of C 2 H 2 and ion F (m/z 65) turned to F1 (m/z 65), when the NEC was higher than 140 eV. Ion C (m/z 103) transformed into TC 1 (75.6 kcal/mol in Figure 4) and ion E2 into TE 3 (70.5 kcal/mol in Figure 5) with higher maximal ΔG' than process of losing CO 2 (ion A2 to TA 3 , 68.6 kcal/mol), both were endothermic reaction. The results were well consistent with the relative abundance of ion C (m/z 103), E (m/z 77)and G (m/z 51) in Figure 6. Therefore, it was further confirmed that the determining factor was the maximal ΔG' in each cleavage pathway for ESI mass.