The present study was to evaluate the effects of Huang Bai, Zhi Mu, Mai Ya, and Xia Ku Cao on hormone using the GT1-7 and GH3 cells. The GT1-7 and GH3 cell lines were incubated with DW; DMSO; and 30, 100, or 300 μg/mL of one of the four extract solutions in serum-free media for 24 hours. The MTT assay was performed to determine the cytotoxicity of the four herbs. The GT1-7 and GH3 cells were incubated in DW, estradiol (GT1-7 only), or noncytotoxic herb solutions in serum-free medium for 24 hours. A quantitative RT-PCR and western blot were performed to measure the GnRH expression in GT1-7 cells and GH expression in GH3 cells. Huang Bai, Zhi Mu, Xia Ku Cao, and Mai Ya inhibited the GnRH mRNA expression in GT1-7 cells, whereas Huang Bai enhanced GH mRNA expression in GH3 cells. Additionally, Xia Ku Cao inhibited GnRH protein expression in GT1-7 cells and Huang Bai promoted GH protein expression in GH3 cells. The findings suggest that Huang Bai can delay puberty by inhibiting GnRH synthesis in the hypothalamus while also accelerating growth by promoting GH synthesis and secretion in the pituitary.