Dehydration is one of the key steps for protein folding. While theoretical analyses suggest that the large decrease in the conformational entropy is associated with protein folding, the folding experiments show rather small decrease in the entropy, which allows us to speculate that release of hydrated water molecules from the peptide entropically contributes to the protein folding. However, no experimental data have reported on the dehydration associated with protein folding. Here, we focused on the volume changes in protein folding to discuss the dehydration and determined the volume changes associated with protein folding of reduced cytochrome c (Cyt c) from the unfolded state to the native state. The equilibrium constant between the unfolded state and native state (K_UN) was determined by a change in the absorption (420 nm) at various pressures between 0.1 and 200 MPa and at various concentrations of denaturant (guanidine hydrochloride) between 3.2 and 4.0 M. Dependence of K_UN on these factors revealed that the volume change at ambient pressure in the absence of denaturant is negative (ΔV_UN = -25(±10) cm³·mol^-1). We also followed pressure dependence of the folding rate (k) to determine the activation volume (ΔV^‡) for the process from the collapsed state (the initial intermediate for the protein folding) to the native state, by using the photo-induced protein folding reaction. The folding rate was followed by a change in the absorption (420 nm) at various pressures between 0.1 and 200 MPa and at various concentrations of the denaturant between 3.2 and 4.0 M. ΔV^‡ at ambient pressure in the absence of denaturant is also negative (-14(±8) cm³·mol^-1) and comparable to ΔV_UN. Such negative volumes can be accounted for by a decrease in volume resulting from the dehydration of hydrophobic groups, primarily the heme group, and the dehydration is mainly induced in the process from the collapsed state to the native state. The present data clearly indicates that dehydration, which increases the entropy of the protein system, compensates for a decrease in the conformational entropy accompanying the formation of the more compact and ordered transition state. We, therefore, propose that the rather small change in the entropy for the folding reaction of Cyt c is due to the dehydration of hydrophobic groups, and dehydration entropically promotes the protein folding reaction.