In order to further understand the deactivation behavior of a NiMo/HZSM-5 catalyst system used for fluid catalytic cracking (FCC) gasoline hydro-upgrading, fresh and coked catalysts after operation for different times-on-stream (TOS) with FCC gasoline as a feedstock were characterized by X-ray diffraction, Fourier transformed infrared (FTIR) spectroscopy, nitrogen adsorption, and temperature-programmed desorption of ammonia, as well as FTIR analysis of adsorbed pyridine. The results showed that the amount, nature, and location of coke formed in the catalysts depended upon TOS. Coke was preferentially formed on the strong acid sites, especially on the strong Lewis acid sites in the pore channels and/or on the external surface of the catalysts, resting with the size of the reactant molecules in FCC gasoline. Coke formation led to increases in the selectivities to C-8, C-9, and C-9(+) aromatics and decreases in the selectivities to benzene and toluene in the aromatics products. Using real FCC gasoline as a feedstock, this work showed that the deactivation behavior of the HZSM-5 zeolite-based catalyst was different from that obtained using model compounds as feedstocks due to the wide size distribution of the hydrocarbon molecules in FCC gasoline and to the complex reaction mechanisms among these components. The results provided some clues for finely tuning the physicochemical properties of the catalyst to further enhance its on-stream stability.