This study aimed to evaluate the effects of phytase super-dosing in the diet of laying quails subjected to different temperatures, their performance, blood biochemistry, and gene expression of the epithelial calcium transporters Calbindin-D28 K and TRPV6. Seven hundred and twenty (720) Japanese quails in the production phase were distributed in a completely randomized design, in a 5 × 3 factorial scheme, with five levels of phytase supplementation (0, 500, 1000, 1500 and 3000 FTU/kg) and three temperatures (24°C, 30°C and 36°C), totaling 15 treatments, and six replicates of eight birds each. The study began at 8 weeks of age and continued for two 21-day cycles, totaling 42 days of experiment. Performance parameters, serum biochemistry, and gene expression of calbindin-D28 K and TRPV6 were measured through real-time PCR. The data were subjected to analysis of variance, Tukey, and regression. Birds kept at 36°C showed lower feed intake than those kept at 24°C and 30°C. At 30°C, birds had lower serum uric acid levels than birds at 36°C, and higher total egg production, egg mass, and Calbindin-D28 K gene expression in the uterus than those at 24°C and 36°C. Birds kept at 24°C had higher serum calcium and phosphorus levels than those kept at 30°C and 36°C. At a supplementation level of 1500 FTU, phytase provided greater eggshell thickness in quails kept at 36°C and greater calbindin-D28 K gene expression in the kidney. Therefore, the use of 1500 FTU of phytase is recommended for Japanese quail exposed to high temperatures, since phytase overdosage has been shown to be effective in mitigating the negative effects of heat stress.