Anticancer drugs in waters may increase potential threats on the future persistence of aquatic organism with their actions on cell death in targeted cell types of neoplasia, such as cyclophosphamide (CP). The potential adverse effects of CP may be exacerbated by the enhance of detected frequency and levels in different waters. Thus, the mechanisms of CP on the Daphnia magna are elucidated in this study. Our results found aspartic acid, citric acid, tyrosine, isoleucine, phenylalanine, phenyl-acetaldehyde, phenyl-acetylglutamine, trans-cinnamate, and methionine as core metabolites were identified in response to CP by LC-MS-based metabolic analysis, implying the disruption of amino, energy and lipid metabolism pathways. Differentially expressed genes by transcriptomic analysis were mainly enriched in the nervous system and lipid metabolism. For the behavior and reproduction performances, CP not only increased the light response and swimming velocity of D. magna, but also delayed the time to first brood, decreased the number of broods, and increased the number of neonates per brood. Overall, oOur findings suggest that CP alters the behavior and reproduction, the metabolites, and transcriptional expression in the nervous system and metabolism pathways and provided some evidence on the mechanisms of CP toxicity in D. magna and perhaps other zooplankton.