This study was carried out to investigate the effects of 1, 25-Dihydroxycholecalciferol (1, 25 (OH)2 D3) (calcitriol) and root hydroalcoholic extract of Withania coagulans (WC) root on immune response and small intestinal morphology of broiler chickens. Treatments were arranged factorially (2×3×2) consisted of a positive control with adequate Ca and a negative control diet (Ca level reduced by 30%), three levels of WC (0, 100 and 200 mg/kg diet), and two levels of 1, 25 (OH)2 D3 (0 and 0.5 g/kg diet). Six hundred male day old Ross 308 broiler chicks were randomly distributed into 60 floor pens, 10 birds each. Each treatment was replicated 5 times (50 birds). Each diet was fed ad libitum to chicks from day one to 42 d of age. Sheep red blood cells (SRBC) test was used as measure of humoral immunity. So, at 25 and 32 days of age chicks were injected to evaluate the primary and secondary antibody response. At 37 days of age, the cutaneous basophil hypersensitivity (CBH) test was used to evaluate cell-mediated immune response. On d 21 and 42, one bird per replicate was killed to determine small intestinal morphology and lymphoid organ weights. The results of humoral immune response showed that reduction of dietary Ca level by 30% and supplementation of WC did not significantly influence total antibody levels. The CBH response showed that supplementation of WC and calcitriol had no significant effect on interdigital skin thickness. At 42 d of age, the weight of spleen significantly reduced by feeding of negative control diet. Also, supplementation of WC at 200 mg/kg significantly increased the weight of thymus at this age. It was found that supplementation of WC at 200 mg/kg resulted in shorter villus length at 42 d of age.