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The present study aimed to determine the effect of different times of supplementation of 25-hydroxycholecalciferol (25(OH)D 3 ) in broiler chickens on the performance, carcass and cuts yield, bone resistance, plasma concentration of 25(OH)D 3 , and expression of the mTOR gene. The treatments were a control diet (CD) supplemented with 3000 IU vitamin D 3 /kg of feed from 1 to 46 d, or the CD + 2760 IU (69 mcg) of 25(OH)D 3 /kg of feed from 1 to 21 d, from 1 to 35 d, or from 1 to 46 d. The period of supplementation of 25(OH)D 3 did not affect the growth performance of broilers, but the breast meat yield was linearly increased in response to increasing days of supplementation ( p < 0.05). Birds supplemented with 25(OH)D 3 at the time of the analysis showed an increase ( p < 0.05) in the plasma concentration of 25(OH)D 3 when compared to non-supplemented birds. The mTOR gene expression ( p < 0.05), and breast protein deposition ( p < 0.05) presented a quadratic response related to the supplementation period of 25(OH)D 3 . The fat content of the breast linearly decreased ( p < 0.05) as the period of supplementation was extended. The results also showed a positive linear correlation between mTOR expression and 25(OH)D 3 plasma levels (r = 0.593; p < 0.05).