Inducible defenses occur in various taxa of prey organisms. Many phytoplankton species, including the freshwater Chlorophyta Tetradesmus spp., can form defensive morphs under the zooplankton-released grazing cues. However, definite statements on the actual mechanisms of inducible defense in phytoplankton are quite deficient. In the present study, the defensive mechanism of Tetradesmus obliquus (formerly known as Scenedesmus obliquus) induced by Daphnia infochemicals was analyzed through multifaceted analyses integrating physiological and biochemical monitoring, transcriptomics, and metabolomics. Results showed that the upregulated genes encoding G protein-coupled receptor and phospholipase C were involved in the perception and transduction of grazing cues, and the transcripts related to extracellular polysaccharides formation and transductions were upregulated. The results provided molecular evidence for the role of extracellular polysaccharides in colony formation. Increased lipid content revealed through metabolomics suggested higher investment in lipid biosynthesis within colonial cells, whereas the downregulation of ribosome-related pathways indicated less allocation of energy and resources to protein biosynthesis. The significant increase in indole-3-acetic acid content indicated that growth was activated under grazing cues. This study provided essential information on the inducible defensive mechanism of Tetradesmus and direct evidence for the intracellular reallocation of resources and energy owing to the defensive colony formation, which had considerable implications for enriching the theory of inducible defenses at the molecular level.Long-term arms races between prey and predator drive the coevolution of both sides (Yoshida et al. 2003;True et al. 2018;Wilson et al. 2018). Preys evolve diversified plastic traits including chemical, morphological, and behavioral changes to reduce predation mortality under strong selective effects of predation (Abrams 2000). These adaptive responses are widespread in organisms from unicellular organisms to large vertebrates, which may not only reduce predation but will also greatly affect food web structure via trophic cascades (Werner and Peacor 2003;Nunes et al. 2016;Suraci et al. 2016). Compared with constitutive defenses which are expressed throughout the lifetime, the defenses that are only produced when facing certain predation pressure are known as inducible defenses. Inducible defenses could be either continuous reaction norms or threshold traits depending on the trade-offs between survival and fecundity or competitive ability (Svennungsen et al. 2011). To some extent, inducible defenses are favored by organisms because of their higher effectiveness and lower costs (Clark and Harvell 1992).Grazing is a main contributor to biomass loss in phytoplankton communities (Calbet 2001). Herbivores remove up to 79% of annual algal primary production in aquatic ecosystems (Cyr and Face 1993). A wide taxa of phytoplankton demonstrate effective defenses (e.g., larger size, col...