Objective To establish and evaluate the CaV1.1-R528H gene knock-in mouse model of thyrotoxic hypokalemic periodic paralysis. Methods Thirty-six 8-week-old male CaV1.1-R528H gene knock-in mice and thirty-six 8-week-old wild-type male C57BL/6J mice were used in this study. Using three-factor two-level 2×2×2 factorial design (the three factors including mutation, thyroxine and insulin, and two levels were with or without), the mice were divided into 8 groups. The thyroxine groups were intraperitoneally injected with levothyroxine in a dose of 350 μg/kg once per day for 12 consecutive days to produce thyrotoxicosis. The insulin groups were intraperitoneally injected with short-acting insulin in a dose of 0.8 U/kg after the last administration of levothyroxine, and the potassium levels of different groups were measured and recorded before (0 min) and after insulin injection (30 min, 60 min).Results (1) Compared with the control group, the following phenomena including irritability, dull coat, increased diet and water intake, and slow body weight gain, were observed in the thyrotoxic mice. Thyroid function tests showed that the levels of T3 and T4 in the thyrotoxic mice were significantly higher than those in the corresponding control mice (P<0.05), and the TSH level was significantly lower than that of the corresponding control mice (P<0.05 ).(2) After administration of insulin or thyroxine alone, the potassium levels in the mutant and wild-type mice were not significantly different. However, after combined administration of thyroxine and insulin, the potassium levels in the mutant group were significantly lower than those in the wild-type mice at 30 min and 60 min (P<0.05 for both). (3) The main effects and interactions: Mutation factor or thyroxine factor alone did not influence on the potassium level, only insulin showed hypokalemic effect (P<0.05). There were interactions between thyroxine and mutation, and between insulin and mutation (P<0.05), but no interaction between thyroxine and insulin. Conclusions (1) A thyrotoxicosis state in mice is successfully developed in this study. (2) An CaV1.1-R528H gene knock-in mouse model of thyrotoxic hypokalemic periodic paralysis is successfully established.