The protective effects of osmolytes on the renaturation of Pelodiscus sinensis brain-type creatine kinase (P-CKB) were determined in this study. The P-CKB gene was cloned and was heterologously expressed in Escherichia coli BL21 (DE3). The purified recombinant protein was subjected to 6 M urea denaturation and further applied with six different osmolytes (glycine, proline, sorbitol, DMSO, betaine, and trehalose). Our results demonstrated that the addition of glycine or DMSO could contribute toward the reactivation of unfolded P-CKB and prevent aggregation. Interestingly, 25 mM glycine was found to be the best concentration to reactivate denatured P-CKB, while high-concentration glycine led to the opposite effect as monitored by a red shift in the intrinsic fluorescence spectra and the aggregation of protein, which probably could be attributed to an additional inactive protein complex that formed during the fast refolding reaction. Docking simulation results showed the osmolyte docking energies to be relatively low and clustering groups were spread on the surface of P-CKB, indicating that osmolytes directly protected the surface of P-CKB. The results in this study could provide a better understanding of structural and functional changes in P-CKB during refolding in addition to the role of osmolytes in heterothermic animals.