Abstract: 【Objective】 To investigate the effect of follicle stimulating hormone（FSH）on the formation of primordial follicles in chicks，which could provide theoretical basis for research on the early development of the ovary in chicks and the improvement of egg-laying performance in laying hens. 【Method】 Forty freshly fledged chicks were randomly divided into test group（FSH）and control group（CK），and were injected intramuscularly with equal amounts of 20 IU/ mL FSH and saline respectively. The chicks were weighed at 0 and 5.5 d after the start of the experiment，and the ovarian tissues were collected at 5.5 d for paraffin sectioning，HE staining and transcriptome sequencing（RNA-Seq）analysis. Differen‐ tially expressed genes（DEGs）were screened by bioinformatics analysis，and GO function annotation analysis and KEGG signal pathway enrichment analysis were performed. The DEGs were uploaded to the STRING database to construct the protein interaction（PPI）network. Finally，the relative gene expression was detected by real-time fluorescence quantitative PCR. 【Result】 Compared with the CK group，there was no significant change in the body weight of the chicks in the FSH group（P>0.05，the same below），and the volume of the ovary increased greatly；HE staining results showed that the ovarian cortex was thicker in the FSH group，the ovarian tissues were denser，the number of primordial follicles was increased and their volume was enlarged and the primordial follicular structure could be clearly seen. There were 617 DEGs in the ovaries of the 2 groups，107 in the FSH group and 510 in the CK group. The analysis of GO function annotation and KEGG signal pathway enrichment showed that the GO function entries annotated by DEGs could be classified into 3 major categories：biological processes，cellular components and molecular functions，and were mainly enriched in the signal pathways such as PI3K-AKT，Wnt/β-catenin，MEK/ERK and MEK1/2/ERK1/2. The results of real-time fluores‐ cence quantitative PCR showed that compared with the CK group，the relative expression of DAZL and PIWIL1 genes in the FSH group was extremely significantly decreased（P<0.01，the same below），the relative expression of SMAD2，ZP1 and CYP17A1 genes was significantly（P<0.05）or extremely significantly increased，and there was no significant change in the relative expression of FSHR genes. 【Conclusion】 FSH may promote the formation of primordial follicles in chicks by affecting several signal pathways related to follicular development，such as granulosa cell proliferation，steroidogenesis and angiogenesis，and its regulation of genes such as CYP17A1，SMAD2 and ZP1 may be one of the main molecular mechanisms to promote primordial folliculogenesis.