Different challenges are being applied in the poultry industry in order to protect animal health and to increase immunity and production. The supplementation of microbial phytase is essential in terms of both reducing the inorganic phytase rate and contributing to the absorption of other minerals. In this study, a newly isolated microbial phytase was added at different concentrations to the diet together with calcium (Ca2+) and available phosphorus (AP), and the effects of this supplementation on intestinal absorption capacity and Ca2+ binding capacity were investigated via morphological measurements and immunohistochemical examination of the duodenum and ileum. For this purpose, 90 Lohmann LSL-White laying hens were divided into three main diet groups: 1. Standard Ca2+ and AP (Ca+AP), 2. Standard Ca2+ and low AP (Ca+low AP), and 3. Low Ca2+ and low AP (low Ca+low AP). These three groups were further divided into three phytase subgroups each (without phytase [Phy-], commercial phytase [CP] and microbial phytase [MP]). At the end of the experiment, animals were euthanized, and duodenum and ileum samples were fixed and processed for histological examination. Villus height, crypt depth, total mucosa thickness, and villus width were measured and villus height: crypt depth ratio and villus absorption area were calculated. Caldesmon expression in the duodenum and ileum was also investigated immunohistochemically. The results indicated that villus height, total mucosa thickness, and villus absorption area increased (p ≤ 0.05) in birds fed with Ca+AP+MP. Stronger caldesmon expression was observed in the MP treated groups. We concluded that MP produced from Bacillus megaterium EBD 9-1 bacterium increases the utilization of Ca2+ and AP and, thus, can have a beneficial role when these macrominerals are used insufficiently. Ca2+, AP, and MP may have positive effects on the intestinal morphology and absorption area when used at optimum amounts.