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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.