The chromatographic behaviors of benzoic acid (BAH) were investigated on a self-made p-tert-butyl-calix [4] arene-1,2-crown-4 (Cx4-4) stationary phase by changing methanol content and pH value of the mobile phase. The results show that hydrophobic interaction is the main interaction in the separation of benzoic acid, moreover, inclusion interaction, pi-pi and hydrogen bonding interactions also play additional roles. A Density Functional Theory (DFT) method with the base set of B3LYP/STO-3G* was employed to explain the interaction between BAH and Cx4-4, and the optimized supramolecular structure (Cx4-4 and BAH), the Gibbs free energy change (delta G) and stabilization energy change (delta E) were obtained. With the assistance of quantum chemistry calculation, the separation mechanism is further discussed. The quantum chemical calculation results were consistent with the retention behavior of BAH on Cx4-4 stationary phase. Finally, by using the self-made Cx4-4 column, a high performance liquid chromatographic (HPLC) method for the analysis of BAH in tuber mustard and vinegar was developed.