Aluminum oxide cluster anions are produced by laser ablation and reacted with n-butane in a fast flow reactor. A reflectron time-of-flight mass spectrometer is used to detect the cluster distribution before and after the reactions. Aluminum oxide clusters Al2O4,6− and Al3O7− can react with n-C4H10 to produce Al2O4,6H− and Al3O7H−, respectively, while cluster Al3O6− reacts with n-C4H10 to produce both the Al3O6H− and Al3O6H2−. The theoretical calculations are performed to study the structures and bonding properties of clusters Al2O4,6− and Al3O6,7− as well as the reaction mechanism of Al2O4− + n-C4H10. The calculated results show that the mononuclear oxygen-centred radicals (O−˙) on Al2O4,6− and Al3O7−, and oxygen-centred biradical on Al3O6− are the active sites responsible for the observed hydrogen atom abstraction reactivity. Furthermore, mechanism investigation of the O−˙ generation in Al3O7− upon O2 molecule adsorption on un-reactive Al3O5− is performed by theoretical calculations.