This study evaluated the effect of 25-hydroxycholecalciferol (25-hydroxyvitamin D3 , 25-OH-D3) on growth rate, femur calcium (Ca) content, and mRNA abundance of intestinal Ca transporters in broiler chickens aged 1–21 days. A total of 420 one-day-old female Arbor Acres broilers were assigned to six treatments. Diet 1 did not contain 25-OH-D3 (0 µg/kg 25-OH-D3); diets 2–6 were based on diet 1, but additionally supplemented with 3.125, 6.25, 12.5, 25, and 50 µg/kg 25-OH-D3. The results showed that the addition of 3.125–50 µg/kg 25-OH-D3 increased body weight gain, feed intake, bone mass, and ash, Ca, and phosphorus content in the femur of 1–21-day-old broilers compared to diet 1 (0 µg/kg 25-OH-D3). Supplementing 6.25–50 µg/kg 25-OH-D3 increased transcript levels of Ca-binding protein D28k (CaBP-D28k) in the duodenum, jejunum, and ileum compared to diet 1. The level of CaBP-D28k mRNA expression in the three intestinal segments increased by 1171-, 2091-, and 46-fold, respectively, after the addition of 50 µg/kg 25-OH-D3. The levels of plasma membrane Ca ATPase 1b (PMCA1b) mRNA in the jejunum and ileum increased after supplementing 12.5–50 µg/kg 25-OH-D3. The abundance of sodium-Ca exchanger 1 (NCX1) mRNA increased in the duodenum after the addition of 12.5 and 50 µg/kg 25-OH-D3. No differences were observed in mRNA levels of CaBP-D28k, PMCA1b, and NCX1 in the jejunum and ileum in broilers fed 12.5–50 µg/kg 25-OH-D3. These data demonstrated that 25-OH-D3 improved the growth rate and promoted transcription of the intestinal Ca transporter gene in broiler chickens aged 1–21 days. The requirement of broilers for 25-OH-D3 was at least 12.5 µg/kg based on gene expression levels of intestinal Ca transporters genes.