Isobutanal is an aliphatic aldehyde which has been extensively studied as an important intermediate in isomerization reactions as well as in astrochemically relevant models in the interstellar medium. Herein, we report on the conformer-specific photoionization and conformational stabilities of isobutanal utilizing one-photon mass-analyzed threshold ionization (MATI) spectroscopy with vacuum ultraviolet (VUV) pulses. The conformational population of isobutanal under different supersonic expansion conditions was explored to identify the conformers, from which their intrinsic photoionizations can be directly elucidated by measuring the VUV-MATI spectrum corresponding to each conformer. The observed MATI spectra could be analyzed through Franck-Condon simulations at the B3LYP/cc-pVTZ level for the isobutanal conformers, trans and gauche, for which the adiabatic ionization energies were precisely determined to be 78 133 ± 3 cm-1 (9.6873 ± 0.0004 eV) and 78 557 ± 3 cm-1 (9.7398 ± 0.0004 eV), respectively. Notably, only the gauche conformer undergoes a unique geometrical change upon ionization, resulting in the progression of the CHO torsional mode in the MATI spectra. Consequently, we determined the conformational stabilities of isobutanal by conformer-specific photoionization, given that the gauche is more stable than the trans by 162 ± 50 cm-1 in the neutral ground state, while the cationic gauche is less stable than the cationic trans by 262 ± 50 cm-1.