Prions containing misfolded prion protein (PrP Sᶜ) can be formed with cofactor molecules using the technique of serial protein misfolding cyclic amplification. However, it remains unknown whether cofactors materially participate in maintaining prion conformation and infectious properties. Here we show that withdrawal of cofactor molecules during serial propagation of purified recombinant prions caused adaptation of PrP Sᶜ structure accompanied by a reduction in specific infectivity of >10 ⁵-fold, to undetectable levels, despite the ability of adapted “protein-only” PrP Sᶜ molecules to self-propagate in vitro. We also report that changing only the cofactor component of a minimal reaction substrate mixture during serial propagation induced major changes in the strain properties of an infectious recombinant prion. Moreover, propagation with only one functional cofactor (phosphatidylethanolamine) induced the conversion of three distinct strains into a single strain with unique infectious properties and PrP Sᶜ structure. Taken together, these results indicate that cofactor molecules can regulate the defining features of mammalian prions: PrP Sᶜ conformation, infectivity, and strain properties. These findings suggest that cofactor molecules likely are integral components of infectious prions.