An experiment was conducted to evaluate phytase supplementation on growth, phytate degradation, and the gene expression of myo-inositol transporters in 21-day old broilers. Ross 308, male broilers (n = 240) were assigned to one of four diets, with 10 pens/diet and six birds/pen from day one to 21. The diets consisted of a negative control (NC) formulated to meet or exceed Ross 308 nutrient requirements, with the exception of calcium (Ca) and available P (avP), which were reduced by 0.16 and 0.15%, respectively. The NC diet was supplemented with 0, 500, 1,500, or 4,500 units/kg of phytase (FTU) to create four experimental diets. On day 21, all birds per pen were euthanized to obtain digesta and tissue samples for phytate degradation and gene expression. Data were analyzed as an analysis of variance using the fit model platform in JMP v 13.0. The model included phytase and significant means were separated using orthogonal linear and quadratic contrasts. Phytase supplementation increased gain (linear, P < 0.05). Phytate (iP6; quadratic, P < 0.05), phytate ester (iP5, iP4, iP3; quadratic, P < 0.05), and inositol (linear, P < 0.05) concentration in the gizzard was influenced by phytase supplementation. Phytate concentration decreased (linear, P < 0.05), iP5 or iP4 concentration increased and then decreased (quadratic, P < 0.05), and inositol concentration increased (quadratic, P < 0.05) in the ileal digesta as phytase supplementation increased in the diet. There was a tendency for the gene expression of the H+-dependent myo-inositol transporter, HMIT, to increase (linear, P < 0.05) in the ileum as phytase dose increased. Gene expression of the sodium-dependent myo-inositol transporter, SMIT2, increased in the jejunum (quadratic, P < 0.05) as phytase dose increased. Intestinal alkaline phosphatase expression increased (linear, P < 0.05) in the ileum as phytase supplementation increased in the diet. The influence of phytase on phytate, phytate esters, and inositol may influence intestinal alkaline phosphatase activity and the gene expression of myo-inositol transporters in the small intestine.