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Rhodotorula gracilis, a red yeast, was shown to produce phytase and the cultural conditions were standardized for phytase production. Permeabilization treatments using detergents, organic solvents, enzymes and physical methods were performed and compared by measuring the phytase activity of whole cells. None of the permeabilization methods except the freeze-thaw method enhanced phytase activity. The enzyme activity of a yeast suspension subjected to 15 cycles of freezing and thawing was 77.26 U g−1 cells. Phytate in feeds is an anti-nutritional factor which complexes with many minerals, rendering it unavailable to monogastric animals. Rhodotorula gracilis subjected to freeze-thawing can be used as a source of phytase in feeds to enhance phosphorus availability.