Effects of the in ovo administration of vitamin D
3
(
D
3
) and 25-hydroxyvitamin D
3
(
25OHD
3
) on broiler intestinal lesion incidence, performance and breast meat yield after a coccidiosis challenge were investigated. On each of 10 incubator tray levels, 10 Ross 708 broiler hatching eggs were randomly assigned to each of the following 5 in ovo injection treatments administrated at 18 d of incubation (
doi
): 1) noninjected; 2) diluent; diluent containing either 3) 2.4 μg D
3
(D
3
), 4) 2.4 μg 25OHD
3
(25OHD
3
), or 5) 2.4 μg D
3
+ 2.4 μg 25OHD
3
(D
3
+25OHD
3
). A 50 μL solution volume was injected into each egg using an Inovoject multi-egg injector. Four male chicks were randomly assigned to each of 80 battery cages in each of 2 rooms. Half of the treatment-replicate cages (8) in each room were challenged with a 20× live coccidial vaccine at 14 d of age (
doa
). One randomly selected bird from each of 4 treatment-replicate cages was scored for coccidiosis lesions before and 2 wk after challenge. Mean BW, BW gain (
BWG)
, feed intake, and feed conversion ratio were determined for all birds from 0 to 14, 15 to 28, and 29 to 41 doa. Carcass weight, and the absolute and relative (% of carcass weight) weights of carcass parts were determined in 3 birds per treatment-replicate cage at 42 doa. Hatchability of live embryonated injected eggs and hatch residue were not affected by treatment. Across challenge treatment, birds in the 25OHD
3
treatment group experienced an increase in BWG between 29 and 41 doa when compared to the D
3
or diluent-injected birds. Furthermore, pectoralis major muscle percentage tended (
P
= 0.059) to increase in birds belonging to the 25OHD
3
treatment in comparison to birds in the D
3
or diluent-injected treatments. These results indicate that regardless of challenge treatment, 2.4 μg of 25OHD
3
may increase the BWG and breast meat yield of birds relative to those that only received an injection of commercial diluent.