The effects of the
in ovo
administration of vitamin D
3
(
D
3
) and its metabolite, 25-hydroxyvitamin D
3
(
25OHD
3
)
,
on the performance, breast meat yield, and inflammatory responses of broilers fed commercial diets were investigated. Live embryonated Ross 708 broiler hatching eggs were randomly assigned to one of the following 5
in ovo
injection treatments at 18 d of incubation: 1) noninjected; 2) diluent; diluent containing 3) 2.4-μg D
3
, 4) 2.4-μg 25OHD
3
, or 5) 2.4-μg D
3
+ 2.4-μg 25OHD
3
. A 50-μL solution volume of each prespecified treatment was injected into each egg using an Inovoject multiegg injector. At hatch, 18 male chicks were randomly assigned to each of 30 floor pens. The BW, BW gain, feed intake, and feed conversion ratio of the birds were determined in each dietary phase. At 14, 28, and 39 d of posthatch age (
doa
), plasma α-1-acid glycoprotein (
AGP
) levels in 1 bird in each of 6 replicate pens per treatment were determined at 14 and 39 doa. The pectoralis major and minor weights of those same birds were also determined. The remaining birds were processed at 43 doa, and the weights of their processing parts were determined. At 39 doa, the
in ovo
injection of 25OHD
3
alone decreased plasma AGP concentrations in comparison with the noninjected, diluent, and D
3
-alone treatment groups. In addition, birds that received 25OHD
3
alone had a greater BW at 42 doa than birds in the noninjected, diluent, and D
3
-alone treatment groups. At 39 and 43 doa, breast meat yield was increased in response to the
in ovo
injection of 25OHD
3
alone in comparison to all other treatments. These results indicate that the
in ovo
injection of 2.4 μg of 25OHD
3
resulted in an improvement in the performance and inflammatory responses of broilers. A reduction in the inflammatory response subsequent to the
in ovo
injection of 2.4 μg of 25OHD
3
may have led to an increase in broiler performance.