Phytase is widely distributed in plants, animal tissues, and microorganisms. Phytases are produced by yeast either naturally [17–20] or by processes based on recombinant DNA technology using different yeast strains [21–26]. Saccharomyces cerevisiae possesses several properties that make it useful for use in biotechnological applications, such as its resistance to high sugar and alcohol concentrations [27] and a high growth rate at increasing temperatures [28]. Phytase have been studied in different yeast strains, such as baker’s yeast [29], and an extracellular acid phytase from S. cerevisiae was recently purified and characterized [30]. Additionally, a phytase-producing yeast strain, identified as S. cerevisiae strain zi (EU188613), was isolated from soil samples from Sao Paulo, Brazil [31]. This strain is potentially a new source of thermostable phytases of commercial interest, particularly because the screen for this yeast was performed using gradual temperature increases. In this study, our aim was to opti- mize the medium composition and culture conditions to maximize the production of phytase by S. cerevisiae strain zi (EU188613) using statistical designs.