Proposed in theory and then their existence confirmed, anion–π interactions have been recognized as new and important non-covalent binding forces. Despite extensive theoretical studies, numerous crystal structural identifications, and a plethora of solution phase investigations, anion–π interaction strengths that are free from complications of condensed-phase environments have not been directly measured in the gas phase. Herein we present a joint photoelectron spectroscopic and theoretical study on this subject, in which tetraoxacalix[2]arene[2]triazine 1, an electron-deficient and cavity self-tunable macrocyclic, was used as a charge-neutral molecular host to probe its interactions with a series of anions with distinctly different shapes and charge states (spherical halides Cl⁻, Br⁻, I⁻, linear thiocyanate SCN⁻, trigonal planar nitrate NO₃⁻, pyramidic iodate IO₃⁻, and tetrahedral sulfate SO₄²⁻). The binding energies of the resultant gaseous 1 : 1 complexes (1·Cl⁻, 1·Br⁻, 1·I⁻, 1·SCN⁻, 1·NO₃⁻, 1·IO₃⁻ and 1·SO₄²⁻) were directly measured experimentally, exhibiting substantial non-covalent interactions with pronounced anion-specific effects. The binding strengths of Cl⁻, NO₃⁻, IO₃⁻ with 1 are found to be strongest among all singly charged anions, amounting to ca. 30 kcal mol⁻¹, but only about 40% of that between 1 and SO₄²⁻. Quantum chemical calculations reveal that all the anions reside in the center of the cavity of 1 with an anion–π binding motif in the complexes' optimized structures, where 1 is seen to be able to self-regulate its cavity structure to accommodate anions of different geometries and three-dimensional shapes. Electron density surface and charge distribution analyses further support anion–π binding formation. The calculated binding energies of the anions and 1 nicely reproduce the experimentally estimated electron binding energy increase. This work illustrates that size-selective photoelectron spectroscopy combined with theoretical calculations represents a powerful technique to probe anion–π interactions and has potential to provide quantitative guest–host molecular binding strengths and unravel fundamental insights in specific anion recognitions.