Eggshell quality deterioration associated with heat stress is a well-known phenomenon. The involvement of the 28-kDa calcium-binding protein (calbindin, CaBP-D28k) localization in this failure is not clearly understood. To test a possible direct effect of ambient temperature on calbindin-D28k localization, 40 White Leghorn laying hens were housed in individual cages and exposed to high ambient temperature (30-33°C) and thermoneutral temperature (20-22°C) which served as a control. Eggshell quality characteristics and immunohistochemical localization of all intestinal segments and eggshell gland calbindin-D28k were performed under both environmental conditions. As expected, egg weight, eggshell thickness, eggshell percentage, and eggshell density were negatively affected by high ambient temperature (P ≤ 0.01). Immunohistochemistry showed that calbindin was localized in the intestinal enterocyte cytoplasm and glandular cell cytoplasm under thermoneutral conditions. However, the calbindin intensity was prominently decreased in ileum, cecum, colon, and eggshell gland under heat stress conditions. Therefore, it could be concluded that calbindin-D28k localization in intestinal segments and eggshell gland is negatively affected by high ambient temperature which might be related to the deterioration of eggshell quality characteristics under heat stress conditions.