Given the widespread use of insecticides in the environment, it is important to perform studies evaluating their potential effects on humans. Organophosphate insecticides, such as chlorpyrifos, are being phased out; however, the use of pyrethroids in household pest control is increasing. While chlorpyrifos is relatively well studied, much less is known about the potential neurotoxicity of cyfluthrin and other pyrethroids. To gain insights into the neurotoxicity of cyfluthrin, we compared and evaluated the toxicity profiles of chlorpyrifos and cyfluthrin in primary human fetal astrocytes. We found that at the same concentrations, cyfluthrin exerts as great as, or greater toxic effects on the growth, survival, and proper functioning of human astrocytes. By using microarray gene expression profiling, we systematically identified and compared the potential molecular targets of chlorpyrifos and cyfluthrin, at a genome-wide scale. We found that chlorpyrifos and cyfluthrin affect a similar number of transcripts. These targets include molecular chaperones, signal transducers, transcriptional regulators, transporters, and those involved in behavior and development. Further computational and biochemical analyses show that cyfluthrin and chlorpyrifos upregulate certain targets of the interferon-gamma and insulin-signaling pathways and that they increase the protein levels of activated extracellular signal-regulated kinase 1/2, a key component of insulin signaling; interleukin 6, a key inflammatory mediator; and glial fibrillary acidic protein, a marker of inflammatory astrocyte activation. These results suggest that inflammatory activation of astrocytes might be an important mechanism underlying neurotoxicity of both chlorpyrifos and cyfluthrin.