The antinutritional property of phytate is the main problem associated with food animals, but phytase-producing probiotic strain could be the best alternative for resolving this problem. In the present study, phytase from probiotic strain Bacillus subtilis P6 was purified to homogeneity, which showed a relative molecular mass of 40 kDa on native and SDS-PAGE. The purification fold of purified phytase was 86.99, with a specific activity of 104.39 U/mg protein. The purified enzyme showed optimum activity at 40 • C and pH 6.0. Semi-metal ions, such as K + and Ca 2+ , slightly enhance the enzyme activity while Cu 2+ , Co 2+ , and Zn 2+ prominently inhibited phytase activity. Purified phytase showed the highest substrate specificity toward its natural substrate phytate and did not hydrolyze other substrates having a phosphate bond. The purified enzyme exhibited strong proteolytic tolerance against trypsin and pepsin. Further, we evaluated the probiotic potential of B. subtilis and noted that it showed excellent resistance toward high acidic and bile concentrations. Subsequently, it revealed exceptional antimicrobial activity, good hydrophobicity, autoaggregation ability, nonhemolytic, and nonlecithinase activity. Additionally, B. subtilis P6 displayed susceptibility against major antibiotics. Owing to the excellent biochemical properties of phytase with notable probiotic characteristics, B. subtilis P6 may offer the possibility of being a promising candidate for the application in animal feed and food supplements. However, the in vivo use of strain secreting phytase needs to be studied in animals for its intended benefits