To study vitamin D3 regulation of β-defensins genes in the different tissues of Silky Fowl, real-time quantitative PCR method was used to analyze the expression of AVBD-1, GAL-1 and AVBD-5 in thymus, duodenum, jejunum, ileum, cecum and bursa F. of Silky Fowl with fed different levels of vitamin D3 (800, 1 600, 3 200 and 6 400 IU/kg). The results showed that three β-defensins gene expressions were found in six tissues of Silky Fowl, and the highest gene expression levels of AVBD-1 and GAL-1 were both in jejunum of Silky Fowl which fed 3 200 IU vitamin D3; the highest gene expression level of AVBD-5 in duodenum of Silky Fowl was in group fed 1 600 IU vitamin D3; in the tissues of Silky Fowl fed 800, 1 600, 3 200 and 6 400 IU vitamin D3 per kg diet, respectively, expression of the three AVBDs genes were directly up-regulated, then rapidly down-regulated. The results indicated that a dose-dependent manner that the gene expression levels in same tissues and vitamin D3 supplementary levels in diet of Silky Fowl was seen, and the suitable level of vitamin D3 could directly up-regulate the gene expression of β-defensins in tissues of Silky Fowl.