Vitamin D2, or ergocalciferol, is a fungal source of vitamin D suitable as a dietary supplement for vegans. Spent brewer's yeast is an inexpensive by‐product of the brewing industry which may be used as a starting raw material for the enrichment of D2 from the precursor ergosterol. Dried brewer's yeast was reconstituted in water and stirred during exposure to ultraviolet light (UV) for an optimized period of time to convert ergosterol to ergocalciferol. After freeze drying, the D2 enriched yeast was mixed with rice hulls and wheat germ oil for SFE extraction of D2. The SFE parameters consisted of an extraction temperature of 40°C, a solvent to feed of 75 (g CO2/g material), and a flow rate of 75 g/min in a 4L extraction vessel. 100 g of material was extracted and separated in a two stage separator (separator 1, 1500 psi, 40°C; separator 2, 750 psi, 40°C). The extraction pressure was experimentally varied (3000, 6000, and 9000 psi) to understand extraction efficiency from the yeast matrix. UV treatment of brewer's yeast led to a D2 enhancement from non‐detectable levels to 3.8 mg/g (152,000 IU/g). The percentage of D2 extracted from the yeast at the extraction pressure of 3000, 6000, and 9000 psi was 55, 35, and 31% of the starting concentration respectively. The D2 concentration was 1.5 fold higher in separator #2 compared to separator #1. SFE extraction of vitamin D2 from UV enhanced yeast is more efficient at lower extraction pressures.