Cytochrome c (Cyt-c) was encapsulated in hollow mesoporous silica spheres as a support for immobilization. Cyt-c was denatured by an aqueous solution of guanidine hydrochloride to increase the flexibility of Cyt-c structure by unfolding and subsequently passed through the mesopores into the hollow spaces by diffusion. After incorporation, the refolding of Cyt-c was carried out by removing the guanidine hydrochloride using dialysis. Considering that the mesopore size was slightly smaller than the native Cyt-c size, Cyt-c was immobilized inside the hollow spheres. Modification of encapsulated Cyt-c with 18-crown-6 showed catalytic activity for the asymmetric oxidation of methyl 4-tolyl sulfoxide. The encapsulated catalyst could be reused to achieve almost the same catalytic activity.