Research on the energy metabolism of various protozoan parasites showed the essentiality and benefits of cholesterol in the cultivation of these organisms. However, not much is known about the energy metabolism of Histomonas meleagridis, although such information is of high importance to improve cultivation of the parasite for advancements in diagnostics, research and vaccine development. By supplementing a serum enriched cultivation medium with cholesterol, numbers of parasites could be doubled in comparison to unsupplemented negative controls. This effect was demonstrated for two different strains of the parasite, at different levels of in vitro-passages and for histomonads under xenic or monoxenic settings. Supplementing medium free of serum with cholesterol, resulted in significant growth of the parasite over 72 h. However, there were differences in growth behaviour in serum free medium between the different histomonad cultures and continuous passaging of the cultures without serum was not possible. Monitoring the bacterial growth of two different co-cultivated E. coli strains in monoxenic histomonad cultures during these experiments showed that there was no significant impact of cholesterol on the bacteria. Therefore, a direct effect of cholesterol on the parasite itself could be demonstrated. The results of these experiments supply new insights into the metabolism of H. meleagridis and it can be concluded that cholesterol is an important component to enhance parasite growth in vitro.