The purpose of this study was to examine the Escherichia coli 6-phytase on performance and egg quality in Hi-Sex laying hens fed phosphorus-deficient diets. A total of 21600 Hi-Sex laying hens (48 week old) were divided in to three treatment groups and each treatment has 6 replicates (n= 1200 birds). The first group fed a basal diet (0.39% AP) as positive control, the second group fed phosphorus-deficient diet (0.26% available phosphorus) as negative control and the third group fed phosphorus-deficient diet ( 0.26% available phosphorus) plus 60 g/ ton feed Escherichia coli 6-phytase (500 FTU/Kg) for 30 days. All hens were fed the same amount of diets being, 112 gram / day during all the experiment period. Egg production, egg weight and egg quality characteristics were measured three times during the experiment at 0 day, 14 days and 30 days. Egg yolk and albumin weights were not influenced by reducing either available phosphorus or phytase supplementation. While; shell weight and shell thickness were decreased by reducing available phosphorus while improved by added phytase to the negative control. Furthermore, egg production, shell quality and feed conversion ratio were improved at the termination of the trial by added E. coli 6-phytase to the negative control. Interestingly, egg yolk cholesterol was decreased by phytase supplementation on the other hand; calcium, phosphorus and fat contents in yolk were increased. In conclusion, the results indicated that Escherichia coli 6-phytase was efficacious in corn-soybean meal-phosphorus deficient diets fed to Hi-sex laying hens and can be used to reduce diet supplementation with inorganic phosphorus and to improve egg shell quality