Staurosporine (STS), a wide-spectrum kinase inhibitor, is widely used in studies of apoptosis in mammalian cells. However, its physiological and mechanistic effects have never been clearly defined in insect cells, and other applications of STS have rarely been reported. The present study reveals the insecticidal activity of STS on larvae of Mythimna separata Walker, and the apoptotic mechanism induced by STS on lepidopteran Sf9 cell lines. We demonstrate that the viability of Sf9 cells is inhibited by STS in a time- and concentration-dependent manner. Intracellular biochemical assays show that STS-induced apoptosis of Sf9 cells coincides with a decrease in the mitochondrial membrane potential, the release of cytochrome c into the cytosol, a significant increase of the Bax/Bcl-2 ratio, and a marked activation of caspase-9 and caspase-3. These results indicate that a mitochondrial-dependent intrinsic pathway contributes to STS induced caspase-3 activation and apoptosis in Sf9 cells which is homologous to the mechanisms in mammalian cells. This study contributes to our understanding of the mechanism of insect cell apoptosis and suggests a possible new application of STS as a potential insecticide against Lepidopteran insect pests in agriculture.