In recent years, harmful algal blooms (HABs) have occurred with increasing frequency in coastal waters worldwide, posing expanding threats to marine ecosystems. Several known dinoflagellate species within the genus Karenia have formed large-scale blooms, resulting in mass mortality of fish and shellfish, substantial economic losses, and severe degradation of fishery resources and marine ecosystem health. However, the toxicity and toxic components of Karenia papilionacea isolated from the Yellow Sea, China, remain poorly understood. In this study, we investigated the acute toxicity of K. papilionacea toward the model fish Oryzias melastigma. Exposure to K. papilionacea caused dose-dependent acute toxicity in O. melastigma. At a cell density of 1.0 × 10⁴ cells mL⁻¹ over 96 hours, 73% of the tested individuals died, and the surviving fish exhibited significantly impaired swimming behavior. Histopathological examination revealed tissue damage in the intestine and gills of exposed fish. To identify the causative toxic compound, we employed a combination of column chromatography, semi-preparative liquid chromatography, high-resolution mass spectrometry, and quantitative nuclear magnetic resonance spectroscopy. A novel toxin was isolated and structurally characterized from this ichthyotoxic dinoflagellate for the first time. These findings provide a foundation for future investigations into the toxicological mechanisms of K. papilionacea on fish.