1α,25-Dihydroxyvitamin D3(VitD3) is the active form of vitamin D, and it regulates gene expression and protein synthesis in mammalian follicle development. However, the function of VitD3in the follicular development of layers remains unclear. This study investigated, through in vivo and in vitro experiments, the effects of VitD3on follicle development and steroid hormone biosynthesis in young layers. In vivo, ninety 18-week-old Hy-Line Brown laying hens were randomly divided into three groups for different treatments of VitD3(0, 10, and 100 μg/kg). VitD3supplementation promoted follicle development, increasing the number of small yellow follicles (SYFs) and large yellow follicles (LYFs) and the thickness of the granulosa layer (GL) of SYFs. Transcriptome analysis revealed that VitD3supplementation altered gene expression in the ovarian steroidogenesis, cholesterol metabolism, and glycerolipid metabolism signaling pathways. Steroid hormone-targeted metabolomics profiling identified 20 steroid hormones altered by VitD3treatment, with 5 being significantly different among the groups. In vitro, it was found that VitD3increased cell proliferation, promoted cell-cycle progression, regulated the expression of cell-cycle-related genes, and inhibited the apoptosis of granulosa cells from pre-hierarchical follicles (phGCs) and theca cells from prehierarchical follicles (phTCs). In addition, the steroid hormone biosynthesis-related genes, estradiol (E2) and progesterone (P4) concentrations, and vitamin D receptor (VDR) expression level was significantly altered by VitD3. Our findings identified that VitD3altered the gene expression related to steroid metabolism and the production of testosterone, estradiol, and progesterone in the pre-hierarchical follicles (PHFs), resulting in positive effects on poultry follicular development.