Cholesterol is exclusively produced by animals and is present in the plasma membrane of all animal cells. In contrast, the membranes of fungi and plants contain other sterols. To explain the exclusive preference of animal cells for cholesterol we propose that cholesterol may have evolved to optimise the activity of a crucial protein found in the plasma membrane of all multicellular animals, namely the Na+,K+-ATPase. To test this hypothesis mirror tree and phylogenetic distribution analyses have been conducted of the Na+,K+-ATPase and 3β-hydroxysterol Δ24-reductase (DHCR24), the last enzyme in the Bloch cholesterol biosynthetic pathway. The results obtained support the hypothesis of a co-evolution of the Na+,K+-ATPase and DHCR24. The evolutionary correlation between DHCR24 and the Na+,K+-ATPase was found to be stronger than between DHCR24 and any other membrane protein investigated. The results obtained, thus, also support the hypothesis that cholesterol evolved together with the Na+,K+-ATPase in multicellular animals to support Na+,K+-ATPase activity.