Abstract: The cell viability and autophagic vacuoles of the beta-cypermethrin treated Sf9 cells (Spodoptera frugiperda cell) were evaluated and the autophagic-related proteins of Sf9 cells was analyzed. 3-(4,5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide(MTT) assay, microscope assay, transmission electron microscope(TEM) assay, monodansylcadaverine(MDC) fluorescent staining assay and western blot(WB) assay were employed for the investigation. Results showed that beta-cypermethrin inhibited the viability of Sf9 cells in both concentration- and time-dependent manners, and the autophagy was observed in beta-cypermethrin-treated cells. Autophagy-related protein LC3-Ⅱ/LC3-Ⅰand Beclin 1 were up-regulated to 201.32% and 136.00% compared to the control group. And protein p62 was down-regulated to 68.13%. It has been demonstrated that beta-cypermethrin has significant cytotoxicity on Sf9 cells and inhibition the cell's ability via the autophagy pathway.