Milk fat was fractionated into liquid (m.p. ⋍ 12 °C), intermediate (m.p. ⋍ 21 °C) and solid (m.p. ⋍ 39 °C) fractions by three different processes—melt crystallization, short-path distillation and supercritical CO2 extraction—and the cholesterol content of these fractions determined. Cholesterol was enriched in the liquid fractions from all three processes, in particular about 80% of the cholesterol being found in the liquid fraction obtained by short-path distillation. The basis of migration of cholesterol into various milk fat fractions was explained by its affinity to various triglycerides (melt crystallization) and by vapour pressure and molecular weight (short-path distillation). It was more complex in the supercritical CO2 extraction process; the interplay of cholesterol affinity toward CO2 and its molar volume, and its vapour pressure enhancement under applied pressure play a role.