Diatomite (DE) has attracted considerable attention owing to its abundance, low cost, and potential for a wide variety of applications. This work reports the development of mesoporous wool-ball-like (WBL) microspheres from natural DE through a simple hydrothermal treatment. We discovered that the presence of cetyltrimethylammonium bromide is a prerequisite for generating monodispersed WBL microspheres. The mechanism for the transformation of pristine DE into mesoporous microspheres through dissolution–recrystallization was clearly investigated. Interestingly, the microspheres exhibited a specific surface area 25–60 times larger than that of the pristine DE. The application of WBL microsphere DE as an effective support for metallic catalysts in the toluene total oxidation reaction was demonstrated.