Abstract The objective of this study was to investigate the effects of increasing dose of a novel hybrid bacterial 6-phytase on apparent total tract digestibility (ATTD) of energy and nutrients, plasma concentrations of free myoinositol, and retention of calcium (Ca) and phosphorus (P). A total of 15 barrows (21.5 ± 1.4 kg BW) were allotted to a triplicate 5 × 5 Latin square design with 5 dietary treatments and 5 periods per each square. Pigs were individually housed in a metabolism crate. Five dietary treatments consisted of 5 levels of phytase-supplemented diets with increasing phytase units (0, 500, 1,000, 1,500, and 2,000 FTU/kg). Diets contained nutrients meeting the requirement except for Ca and P. Degree of deficient Ca and P content in the NC diet were 0.22 and 0.17%, respectively. Each period lasted 10 days consisting of a 5-d adaptation period and 5-d collection period for total collection. Data were analyzed using orthogonal polynomial contrasts using the Proc MIXED and optimal phytase levels were determined using the NLMIXED of SAS. The increasing phytase supplementation linearly increased (P < 0.05) average daily gain (651 to 695 g/d) and tended to linearly increase (P = 0.072) gain to feed ratio (0.53 to 0.56). Increasing phytase supplementation linearly increased (P < 0.01) ATTD of Ca and P and retention of Ca and P and also showed quadratic effects (P < 0.01). Increasing phytase supplementation linearly increased (P < 0.01) myoinositol content in plasma and also showed quadratic effects (P < 0.05). Broken line analyses showed that ATTD of Ca (P < 0.01) and retention of Ca (P < 0.01) were increased until phytase supplementation were increased (79.4 to 92.1% and 77.4 to 91.4%) from 0 to 671 FTU/kg (SE = 67; P < 0.01) and 0 to 668 FTU/kg (SE = 72; P < 0.01), respectively. Broken line analyses showed that ATTD of P (P < 0.01) and retention of P (P < 0.01) were increased (59.0 to 80.1% and 55.7 to 78.2%) until phytase contents were increased from 0 to 783 FTU/kg (SE = 91; P < 0.01) and 0 to 776 FTU/kg (SE = 76; P < 0.01), respectively. The broken line analyses showed that myoinositol contents in plasma (P < 0.01) were increased (13.2 to 23.2 µmol/L) until phytase supplementation were increased from 0 to 624 FTU/kg (SE = 174; P < 0.01). In conclusion, increasing phytase supplementation up to 2,000 FTU/kg feed improved ATTD of Ca and P, retention of Ca and P, complete hydrolysis of phytate, and growth performance of pigs compared with a P-deficient diet. Phytase supplementation at a range of 600 to 800 FTU/kg feeds provided the optimal improvement of Ca, P, and phytate utilization.