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This study was carried out to investigate the effects of 1, 25-Dihydroxycholecalciferol (1, 25 (OH)2 D3) and root hydroalcoholic extract of Withania somnifera (WS) root on performance, mineral retention, bone mineralisation and mechanical characteristics of broiler chicks. 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 WS (0, 75 and 150 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. On d 21 and 42, one bird per replicate was killed and left tibia removed. Dietary treatments did not influence feed intake and feed conversion efficiency. The maximum body weight gain (2475.75 g) was noted in group fed adequate dietary Ca supplemented with 75 mg/kg WS. The retention of Ca and P were significantly higher at lower level of dietary Ca. Supplementation of 150 mg/kg WS significantly improved dietary Ca retention in birds received a negative control (83.09%) compared to those consumed positive control diet (66.35%). Regardless of dietary Ca level, in birds fed 75 mg/kg WS, addition of 0.5 g/kg 1, 25 (OH)2 D3 significantly improved Ca retention. On d 21, Birds received WS had significantly higher tibia Ca compared to those of control group. Whereas, no significant effects was noted at 42 d. Birds fed on negative control diet supplemented with 75 mg/kg WS and 0.5 g/kg 1, 25 (OH)2 D3 displayed similar tibia Ca compared to those fed only 150 mg/kg WS. Supplementation of 1, 25 (OH)2 D3 in diet significantly increased tibia Ca at 42 d of age. Tibia shear force and stiffness significantly increased by supplementation of Withania somnifera. The present study indicated that Withania somnifera supplementation exerts positive effects on Ca retention, bone calcification and mechanical properties without adverse effects on performance. Also, synergistic effects of withania Somnifera and 1, 25 (OH)2 D3 was observed on Ca retention and bone calcification.