Mesoporous smectite-like materials containing cobalt, SM(Co), were synthesized by a hydrothermal method under different conditions. The materials were used as catalysts for hydrodesulfurization (HDS) of thiophene. The SM(Co) sample is a new type of active HDS catalyst. The activity depends on the synthetic conditions used, but the product distribution does not change so much, giving trans-2-butene, cis-2-butene, and 1-butene with a minor product of n-butane. The high HDS activity of SM(Co) samples may be explained by higher degrees of cobalt dispersion due to the method of preparation used. In addition, the treatment of SM(Co) samples with ammonia solution is effective in enhancing their HDS activities, in particular for originally less active ones. The treatment removes Na+ species involved and this may suppress structural changes of SM(Co) samples by HDS reactions. The ammonia-treated samples maintain high surface area and include much lower concentrations of Na+ species, which would have a negative effect on the activity, and so they indicate enhanced HDS activity.