Objective · To explore the effects of inhibiting thyroid transcription factor 1 (Ttf1) expression on Kiss1 and gonadotropin-releasing hormone (GnRH) expressions in neuronal cells and hypothalamic arcuate nucleus (ARC) of female rats. Methods · Three shRNAs (TTF1-shRNA1, TTF1-shRNA2, and TTF1-shRNA3) interference sequences were designed for rat Ttf1 gene and were constructed into lentiviral vectors. The efficiency of LV-TTF1-shRNA was detected by real-time PCR and Western blotting in neuronal cell line ND7-23. And the expressions of Kiss1 and GnRH were tested 72 h after transfection. Moreover, LV-TTF1-shRNA or the blank lentiviral vector was injected into 21-day female rats’ ARC. Kiss1 and GnRH expressions were detected at the juvenile (25-day), early puberty (35-day) and adult (42-day) stages. Meanwhile, vaginal opening time of the rats was recorded. Results · Seventy-two hours after lentivirus transfection in ND7-23 cells, the mRNA and protein expressions of Ttf1 decreased significantly in LV-TTF1-shRNA2 group and LV-TTF1-shRNA3 group (all P<0.05), particularly in LV-TTF1-shRNA3 group. The expressions of Kiss1 and GnRH mRNA reduced significantly after LV-TTF1-shRNA3 being transfected in ND7-23 cells (both P<0.05). After injection of LV-TTF1-shRNA3 in female rats’ ARC, mRNA abundance of Kiss1 and GnRH showed evident reduction at 25-day, 35-day and 42-day stages (all P<0.05). And KISS1 protein reduced significantly at 35-day and 42-day stages. Female rats transfected with LV-TTF1-shRNA3 showed delayed vaginal opening. Conclusion · Inhibition of Ttf1 gene expression not only significantly reduces the expression of Kiss1 and GnRH in neuronal cells and female rats’ ARC, but also causes female rats vaginal opening delay.