Prion diseases are a group of fatal neurodegenerative illnesses, resulting from the conformational conversion of the cellular prion protein (PrPC) into a misfolded form (PrPSc). The formation of neurotoxic soluble prion protein oligomer (PrPO) is regarded as a key step in the development of prion diseases. About 10%–15% of human prion diseases are caused by mutations in the prion protein gene; however, the underlying molecular mechanisms remain unclear. In the present work, we compared the biophysical properties of wild-type (WT) human prion protein 91-231 (WT HuPrP91-231) and its disease-associated variants (P105L, D178N, V203I, and Q212P) using several biophysical techniques. In comparison with WT HuPrPC, the Q212P and D178N variants possessed greatly increased conversion propensities of PrPC into PrPO, while the V203I variant had dramatically decreased conversion propensity. The P105L variant displayed a similar conversion propensity to WT HuPrPC. Guanidine hydrochloride-induced unfolding experiments ranked the thermodynamic stabilities of these proteins as Q212P < D178N < WT ≈ P105L < V203I. It was thus suggested that the conversion propensities of the prion proteins are closely associated with their thermodynamic stabilities. Furthermore, structural comparison illustrated that Q212P, D178N, and V203I variants underwent larger structural changes compared with WT HuPrPC, while the P105L variant adopted a similar structure to the WT HuPrPC. The mutation-induced structural perturbations might change the thermodynamic stabilities of the HuPrPC variants, and correspondingly alter the conversion propensities for these prion proteins. Our results extend the mechanistic understanding of prion pathogenesis, and lay the basis for the prevention and treatment of prion diseases.