Vacuum ultraviolet photon-induced ionization and dissociation of isoleucine are investi-gated with synchrotron radiation photoionization mass spectroscopy and theoretical cal-culations. The main fragment ions at m/z=86, 75, 74, 69, 57, 46, 45, 44, 41, 30, 28, and 18 from isoleucine are observed in the mass spectrum at the photon energy of 13 eV. From the photoionization e±ciency curves, appearance energies for the principal fragment ions C5H12N+ (m/z=86)、C2H5NO4+ (m/z=75)、C5H9+ (m/z=69)、C4H9+(m/z=57), and CH4N+(m/z=30) are determined to be 8.84±0.07, 9.25±0.06, 10.20±0.12, 9.25±0.10, and 11.05±0.07 eV, respectively, and possible formation pathways are established in detail by the calculations at the B3LYP/6-31++G(d, p) levels. These proposed channels include simple bond cleavage reactions as well as reactions involving intermediates and transition structures. The experimental and computational appearance energies or barriers are in good agreement.