Effects of varying dosages (0.6 to 4.8 μg/egg) of vitamin D3 (D3) and 25-hydroxycholecalciferol (25OHD3), administrated by in ovo (amniotic) injection, on various embryonic and post-hatch physiological variables in Ross 708 broilers were investigated. The in ovo injection of 1.2 to 4.8 μg of both D3 and 25OHD3 increased serum 25OHD3 concentrations in broiler embryos in comparison to non-injected and diluent-injected control groups. Furthermore, the effects of 2.4 μg of D3 and 25OHD3 alone and in combination on the performance, meat yield and quality, small intestine morphology, and immunity of the broilers fed a diet restricted in calcium and phosphorous (ReCaP) content by 20% throughout grow out, period were investigated. In comparison to the in ovo injection of diluent or the combination of D3 and 25OHD3, the in ovo injection of 2.4 μg of 25OHD3 increased breast meat yield at 14 and 40 days of age (doa). In addition, Serum IgM increased in response to the in ovo injection of 25OHD3 at 14 doa. Furthermore, increases in villus length VL to crypt depth ratio (RVC) at both 14 and 40 doa in response to the injection of 25OHD3 alone were observed. The improvements in these observed factors may be due to an improvement in small intestine morphology in response to the in ovo injection of 25OHD3 alone. Lastly, the effects of 2.4 μg of D3 and 25OHD3 alone or in combination on the performance, meat yield and quality, small intestine morphology, and immunity of Ross 708 broilers after being subjected to a coccidiosis challenge at 14 doa were investigated. The in ovo injection of 25OHD3 alone increased broiler BW gain from 29 to 41 doa in comparison to those injected with diluent or D3 alone. Furthermore, the RVC of birds increased before and 2 weeks after a coccidiosis challenge when in ovo-injected with 25OHD3 alone in comparison to being injected with diluent or D3 alone. These results indicate that 25OHD3 alone may improve small intestine morphology, growth performance, and increase meat yield and antibody production of broilers subjected to a coccidiosis infection.