Effects of the in ovo administration of vitamin D3 (D3) and 25-hydroxyvitamin D3 (25OHD3) 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 D3 (D3), 4) 2.4 μg 25OHD3 (25OHD3), or 5) 2.4 μg D3 + 2.4 μg 25OHD3 (D3+25OHD3). 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 25OHD3 treatment group experienced an increase in BWG between 29 and 41 doa when compared to the D3 or diluent-injected birds. Furthermore, pectoralis major muscle percentage tended (P = 0.059) to increase in birds belonging to the 25OHD3 treatment in comparison to birds in the D3 or diluent-injected treatments. These results indicate that regardless of challenge treatment, 2.4 μg of 25OHD3 may increase the BWG and breast meat yield of birds relative to those that only received an injection of commercial diluent.