In view of the wide application of fluoroquinolones (FQs), a group of broad-spectrum synthetic antibacterial agents, and their large ingress into the environment, the toxic effects on non-target organisms caused by FQs have received great attention. In this study, we used zebrafish embryo as a model, measured the general toxic effects of norfloxacin, a commonly used FQs, and investigated the effects of norfloxacin on the neurodevelopment of zebrafish embryos. Our data showed that norfloxacin significantly inhibited the hatching rate of zebrafish embryos, and increased the mortality and malformation rate of the embryos. To discuss the developmental neurotoxicity of norfloxacin, we measured the expression of several stem cell and neuron lineage markers in the zebrafish embryos. We found that norfloxacin exposure inhibited the expression of GFAP (glial cell marker), and enhanced the expression of Sox 2 (stem cell marker) and Eno2 (mature neuron marker). By measuring the level of active Caspase 3 and the expression ratio of Bax to Bcl2, we discovered that norfloxacin induced obvious cell apoptosis in the brain of zebrafish embryos. To explore the mechanism of the developmental neurotoxic effects of norfloxacin, we applied MK-801, a non-competitive NMDA receptors antagonist, to block the actions of NMDA receptors. The results indicated that MK-801 could rescue the upregulated cell apoptosis and disrupted balance of neuro-glial differentiation induced by norfloxacin in the brain of zebrafish embryos. Our results suggest that the activation of NMDA receptors mediates the developmental neurotoxicity of norfloxacin.