Light is a key regulatory factor for photosynthesis and metabolism of microalgae. The diatom Phaeodactylum tricornutum is capable of exhibiting metabolic flexibility in response to light fluctuations. However, the metabolic switching and underlying molecular mechanisms upon illumination transitions remain poorly understood for this industrially relevant marine alga. To address these, the physiochemical and molecular responses of P. tricornutum upon high light (HL) and recovery (HLR) were probed. Upon HL, P. tricornutum exhibited quick responses, including decreases in cell division, major light harvesting pigments (e.g., chlorophyll a, β-carotene, and fucoxanthin), chloroplastidic membrane lipids (e.g., monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and sulfoquinovosyldiacylglycerol), and long-chain polyunsaturated fatty acids (e.g., C20:5), as well as increases in carbohydrates and neutral lipids particularly triacylglycerol. During HLR stage when the stress was remove