This study assessed the effect of limestone particle size and microbial phytase incorporation on the fate of phosphorus (P) and calcium (Ca) along the gastrointestinal tract in 72 laying hens. Four experimental diets were formulated according to a 2 × 2 factorial arrangement to evaluate the effect of two coarse limestone (CL) inclusion. This included a mix (MIX) of 75% CL (2 − 4 mm) and 25% fine particles (FL, <0.5 mm) or 100% FL, in two different basal diets formulated without (MIX0 and FL0) or with 300 FTU of microbial phytase/kg (MIX300 and FL300). Contents of the crop, gizzard, duodenum, jejunum and ileum were collected to determine the mean retention time (MRT) of dry matter (DM), the recovery rate of Ca and P in each segment of the gastrointestinal tract and the apparent fractional digestibility coefficient (AD) of Ca and P in each intestinal segment. In hens fed FL, microbial phytase decreased the MRT of DM along the intestine (p < 0.05). In the crop and the gizzard, Ca recovery increased with MIX incorporation to a greater extent in hens fed without microbial phytase (p < 0.05). The mixed particle size incorporation decreased absorption kinetics of Ca in hens fed microbial phytase. The AD of P and the absorption kinetics of P were significantly decreased in hens receiving FL300, probably due to complex formation between Ca and phytic acid. This study showed that coarse limestone particles incorporation improved mineral utilisation along the digestive tract.