Vitamin D plays an essential role in regulating the bone formation and mineral balance in chickens and it is also related to functional development of the small intestine in rats. This study compared the effects of cholecalciferol and 1,25-dihydroxycholecalciferol on gene expression of vitamin D receptor, intestinal enzymes and nutrient transporters in chickens in the starter phase (1 to 21 d). A total of 1,008 1-d-old male Cobb chicks were used, distributed in a completely randomized factorial design 2 × 3 (2 sources of vitamin D₃: D₃ and 1,25(OH)₂D₃ x 3 levels: 200; 950; 1,700 IU/kg feed) with 6 replicates and 28 birds each one. Relative mRNA abundance of vitamin D receptor (VDR), aminopeptidase N (APN), maltase, sucrase-isomaltase (SI) complex, vitamin D-1-α-hydroxylase, sodium/glucose cotransporter 1 (SGLT1), glucose transporter 2 (GLUT2), peptide transporter 1 (PEPT1), sodium/phosphate cotransporter type IIb (NaPi-IIb), plasma membrane Ca²⁺ ATPase (PMCA-1b) were assayed using real-time PCR. All data were analyzed by SAS GLM and differences among means were separated using Tukey's multiple range tests. There was an interaction (P < 0.05) between sources and levels of vitamin D₃ only for mRNA abundance of SI. The mRNA abundance of maltase, NAPi-IIb, PMCA-1b, PEPT1 and GLUT2 were higher (P < 0.05) in jejunal mucosa of animals fed with vitamin D₃, while the mRNA abundance of 1-α-hydroxylase and SGLT1 were lower (P < 0.05) in these animals. The levels of vitamin D (independently of source) affected the mRNA abundance of 1-α-hydroxylase, PEPT1 and SGLT1. The mRNA abundance of 1-α-hydroxylase in jejunal mucosa of animals fed with 200 IU/kg feed of vitamin D was higher (P < 0.05) compared with 950 IU/kg feed. The mRNA abundance of PEPT1 was higher (P < 0.05) with 200 IU/kg feed of vitamin D compared with other levels, while the mRNA abundance of SGLT1 was higher (P < 0.05) in animals fed with 200 IU/kg feed of vitamin D compared with 1,700 IU/kg feed. It can be concluded that vitamin D and 1,25(OH)₂D₃ can differently influence the gene expression of intestinal enzymes and nutrient transporters in chicken.