Prey are under selection to minimize predation losses. In aquatic environments, many prey use chemical cues released by predators, which initiate predator avoidance. A prominent example of behavioral predator-avoidance constitutes diel vertical migration (DVM) in the freshwater microcrustacean Daphnia spp., which is induced by chemical cues (kairomones) released by planktivorous fish. In a bioassay-guided approach using liquid chromatography and mass spectrometry, we identified the kairomone from fish incubation water as 5α-cyprinol sulfate inducing DVM in Daphnia at picomolar concentrations. The role of 5α-cyprinol sulfate in lipid digestion in fish explains why from an evolutionary perspective fish has not stopped releasing 5α-cyprinol sulfate despite the disadvantages for the releaser. The identification of the DVM-inducing kairomone enables investigating its spatial and temporal distribution and the underlying molecular mechanism of its perception. Furthermore, it allows to test if fish-mediated inducible defenses in other aquatic invertebrates are triggered by the same compound.