Objective To observe the protective effect of the traditional Chinese medicine prescription, Jiu Nao Yi Zhi water extract, on human neuroblastoma SH-SY5Y cell line, its effect on expression of insulin signal transduction pathway, and to explore the related mechanisms. Methods SH-SY5Y cells cultured in vitro, were divided into control group, Jiu Nao Yi Zhi No.1 prescription group and No.3 prescription group. The doses were 0.0625 mg/mL, 0.125 mg/mL, 0.25 mg/mL, 0.5 mg/mL and 1 mg/mL. The thiazolyl blue (MTT) metabolic rate of each group was determined. The dose of 0.125 mg/mL was chosen for cell immunofluorescence analysis, and to observe the expression of insulin receptor substrates-1(IRS-1), cAMP response element binding protein (CREB), and the factors of insulin signal transduction pathway. Results Compared with the control group, MTT metabolic rates of the Jiu Nao Yi Zhi groups were significantly increased (P<0.05), and the cell morphology was much better in those groups, cell body more plump, well-adherent and neurite extensions were observed. The expressions of IRS-1 and CREB were higher than that in the control group.Conclusions The traditional Chinese medicine prescription Jiu Nao Yi Zhi water extract can protect neurons by promoting nerve cell growth, and improving the expression of IRS-1 and CREB, the factors of insulin signal transduction pathway.