Three experiments were conducted to evaluate the effects of 25-hydroxycholecalciferol (25-OH-D(3)) on the growth performance, small intestinal morphology, and immune response of broiler chickens. In experiment 1, 25-OH-D(3) neither increased nor decreased weight gain and feed efficiency compared with the controls during the 39-d feeding period. Birds fed 25-OH-D(3) exhibited numerically higher phagocytosis (45%) than the controls (35%). In experiment 2, chicks were fed diets similar to those used in experiment 1 and were killed at 7, 14, 21, 28, and 35 d of age to determine the relative weight and histology of the small intestine. The relative weight of the small intestine from birds fed 25-OH-D(3) was numerically lower (P < 0.1) at 7 d of age. It was found that 25-OH-D(3) consistently resulted in longer (P < 0.05) villus length of the duodenum in 21- and 28-d-old birds and of the jejunum in 14- and 28-d-old birds. Shorter (P < 0.05) crypt depth was observed in the duodenum at 14 d of age and in the jejunum at 21 and 28 d of age. A higher (P < 0.05) ratio of villus length to crypt depth was also observed in the duodenum and jejunum at 14, 21, and 28 d of age of birds fed 25-OH-D(3). The thickness of muscle layer increased in the duodenum at 14, 28, and 35 d of age in birds fed 25-OH-D(3). In experiment 3, birds were orally challenged with either Luria-Bertani broth or Salmonella Typhimurium E29 at 7 and 14 d of age. Uninfected birds fed 25-OH-D(3) had lower total serum IgA at 14 d of age and lower total serum IgG at 21 d of age. However, infected birds fed 25-OH-D(3) produced higher (P < 0.1) total serum IgG at 21 d of age. The results of this study suggest that supplemental 25-OH-D(3) improves small intestinal morphology and protective humoral immunity to infection.