Abstract: α-Hydroxy-3,6-diene compounds can be synthesized by the nickel-catalyzed carboalkenylation of 1,3-diene with aldehydes and alkenylzirconium reagents. 1,4-Diene structure of α-hydroxy-3,6-diene compounds is a common structural unit in a variety of bioactive molecules, which has been widely used in natural products and can be transformed into diverse polyene structures, and also can be used as a key intermediate for organic synthesis. Mass spectrometric characterization of these α-hydroxy-3,6-diene compounds presents unique ion of M-17⁺. A series of α-hydroxy-3,6-diene compounds, including aroma-α-hydroxy-3,6-diene compounds (Ⅰ) and aliphatic α-hydroxy-3,6-diene compounds (Ⅱ), showed special M-17⁺ during the measurement by atmospheric pressure chemical ionization time-of-flight mass spectrometry (APCI-TOF MS), which might be the fact that these compounds contained the hydroxyl groups and the dehydroxylation process were affected by the unsaturated bond. The results showed that aroma-α-hydroxy-3,6-diene compounds could directly lose one hydroxyl to form M-OH⁺ ions due to the conjugated π bond, while aliphatic α-hydroxy-3,6-diene compounds formed the dehydration and protonation ion M-H₂O+H⁺. It was notably that aroma-α-hydroxy-3,6-diene compounds tended to form the dehydration and protonation ions if they could form conjugated structure by adding protons after dehydration. It was difficult to determine the correct fragmentation pathway of compounds only by simple inference of mass spectrometry. Combined with density functional theory (DFT) calculations, positive ion mode of high resolution mass spectrometry and tandem mass spectrometry was adopted to demonstrate the possible existence of Ⅰ and Ⅱ processes of the nine compounds. It was found that the prediction of a few compound structures did not agree with the actual pyrolysis process. The aforesaid results indicated that gas phase ions in mass spectrometry tended to generate stable structures with low electronic and thermal energies, and the position of unsaturated bonds affected the dehydroxylation process. Simple collision induced dissociation (CID) pyrolysis derivation was applicable for most α-hydroxy-3,6-diene compounds, but ionization process of some special compounds was unreasonable and needed theoretical evidence. Therefore, DFT calculation results was used to support the mass spectrometric process. These results are helpful for accurate identification of the structures of α-hydroxy-3,6-diene compounds or similar compounds, and provide theoretical basis and guidance for the ion writing specification in MS spectra and the fragmentation pathways of these compounds.