Abstract: The effects of pH value and protease in broiler gastro-intestine on Aspergillus niger phytase activity and the ability of phytase to survive in broiler proventriculus, gizzard and small intestine were studied in vitro. The results show that the phytase optimum pH value is 5.5 and is susceptible to broiler gastrointestinal pH and protease. When the phytase incubation at the pH which is higher or lower than its optimum pH5.5， the phytase activities decreased rapidly and most of phytase activity disappeared during 30 min. Following incubation at pH3.0 and pH6.0 in the presence of pepsin and pancreatin respectively， more phytase activity disappeared. Phytase is more susceptible to pancreatin than that to pepsin. 42%， 40% and 49% of phytase were survived on 14， 28 and 42 days broiler proventriculus and gizzard respectively and about 19％of phytase survived in small intestine of broiler in different ages in vitro. The phytase is more stable in proventriculus and gizzard than that in small intestine， where pH and pancreatin is not favor to phytase. The results suggest that phytase is susceptible to pH and protease in broiler gastrointestinal tract， which may be the main reason why only a small part of inorganic P could be replaced by phytase in broiler diet.