〔Abstract〕 Objective To investigate the effect of the hypothalamic mammalian target of rapamycin (mTOR) signaling pathway on puberty onset in female rats and the possible mechanism of the mTOR signaling pathway. Methods Clean grade female Sprague Dawley (SD) rats were selected and randomly divided into normal (N) group, dimethyl sulfoxide (DMSO) group, and rapamycin (Rapa) group. At the age of 23 days, lateral ventricular catheterization was performed on rats in the DMSO and Rapa groups. At the age of 29 to 35 days, rats in the Rapa group were injected with Rapa into the lateral ventricle, while rats in the DMSO group were injected with an equal amount of DMSO into the lateral ventricle. Rats in the N group received no intervention. The rats were weighed daily and the opening of the vulva was observed from the age of 28 days. On the morning of 35 days old, rats in each group were killed 2 hours after the last lateral ventricular injection. Changes in body mass, opening time of vaginal, development of uterine and ovarian, serum luteinizing hormone (LH), follicle stimulating hormone (FSH), estradiol (E2) levels, and hypothalamic mTOR signaling pathway related genes and protein expression in each group of rats were analyzed. Results After lateral ventricle injection of Rapa, compared with the N group and DMSO group, the expression levels of p-mTOR and p-S6K1 proteins in the hypothalamus in Rapa group rats decreased. The Rapa group rats showed delayed opening of the vaginal, and the serum levels of LH and E2, body mass, uterine wet weight, ovarian wet weight, uterine index, ovarian index and expression level of neuropeptide Y (NPY) gene were significantly higher in Rapa group rats (P < 0.05). Conclusion Inhibiting the mTOR signaling pathway in the hypothalamus through lateral ventricular injection of Rapa can delay the onset of puberty in female rats, and its mechanism may be related to the influence of the mTOR signaling pathway on the expression level of NPY in the hypothalamus.