Imidacloprid (IMI) and acetamiprid (ACE) belong to a new class of neonicotinoid pesticides, which are used widely in the world. They act as agonists on nicotinic acetylcholine receptors (nAChRs) and their high toxicities to insects are well studied, but their effects on mammalian nAChRs remain to be elucidated. Many types of nAChRs are important for not only neurotransmission, but also immune system and especially normal brain development. Previously we reported that ACE, IMI, and nicotine exert similar excitatory effects on rat cerebellar neurons (PLoS ONE 2012). In 2013, the European Food Safety Authority (EFSA) considered developmental neurotoxicity of these two neonicotinoids and recommended more stringent regulations. In this study, we examined gene expressions of three independent sets of rat cerebellar cells cultured for two weeks in the absence or presence of IMI, ACE, or NIC at concentrations of 1μM by DNA microarray (Agilent Whole Rat Genome Ver.3.0) and analytical software (Gene Spring GX, Agilent). From a total of 30,367 DNA probes (26,930 gene RNAs) in the DNA array, about 25,000 mRNA transcripts were detected. Between IMI, ACE, or nicotine-treated cultures versus control (C), 247 transcripts (IMI vs. C), 175 transcripts (ACE vs. C), or 100 transcripts (NIC vs. C) showed statistically significant differences (moderate T test, P<0.05) at least 1.5 fold, which include important genes for brain development. Microarray data for selected genes were confirmed by quantitative real-time PCR. The results suggest that neonicotinoids disrupt gene expressions in the developing brain, and are possible causal factors for developmental disorders.