Atrazine is an extensively used herbicide in agricultural and residential areas of the U.S. and routinely detected in many surface and ground waters. This study evaluated the effects of atrazine on the toxicity of selected organophosphate insecticides and gene expression using fourth-instar larvae of the aquatic midge, Chironomus tentans. Atrazine alone up to 1000 µg/L did not show significant toxicity. However, atrazine concentrations from 1 to 1,000 µg/L significantly enhanced the toxicity of dimethoate, disulfoton, or demeton-S-methyl when in combination. Atrazine at 10 µg/L in combination with omethoate significantly decreased insecticide toxicity. Increased toxicity of dimethoate, disulfoton, and demeton-S-methyl in binary combination with atrazine correlated to increased acetylcholinesterase inhibition. Cytochrome P450-dependent O-deethylation activity in atrazine-treated midges was 1.5-fold higher than that in the controls. Cytochrome P450 induction by atrazine may lead to increasing organophosphate toxicity by enhancing oxidative activation of the insecticides into oxon or sulfoxide analogs with increased anticholinesterase activity. To better understand the molecular basis of atrazine’s effects on midges, we used a toxicogenomics approach known as restriction fragment differential display (RFDD)-PCR to systematically compare gene expression profiles between atrazine-treated and –untreated midges. Fifty-six up- and 60 down-regulated genes were tentatively identified in response to atrazine exposure. Atrazine appears to affect the expression of many genes either toxicologically or physiologically important to midges. This study is expected to provide insights into the risk assessment for atrazine and identify atrazine-specific marker genes that can be potentially used to evaluate non-target effects of atrazine and aquatic environmental health.