BackgroundLight is a key regulatory factor for photosynthesis and metabolism of microalgae. The diatomPhaeodactylum tricornutumis 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 ofP. tricornutumupon high light (HL) and recovery (HLR) were probed.ResultsUpon HL,P. tricornutumexhibited quick responses, including decreases in cell division, major light harvesting pigments (e.g., chlorophylla,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 removed, these physiochemical phenotypes were generally recovered, indicative of a rapid and reversible changes ofP. tricornutumto cope with illumination transitions for survival and growth. Through the integrated analysis with time-resolved transcriptomics, we revealed the transcriptional control of photosynthesis and carbon metabolism inP. tricornutumresponding to HL, which could be reversed more or less during the HLR stage. Furthermore, we highlighted key enzymes involved in carotenoid biosynthesis and lipid metabolism ofP. tricornutumand identified monooxygenases putatively responsible for catalyzing the ketolation step towards fucoxanthin synthesis from neoxanthin.ConclusionsThe detailed profiling of physiochemical and transcriptional responses ofP. tricornutumto HL-HLR treatments advances our understanding on the adaption of the alga to illumination transitions and provides new insights into engineering of the alga for improved production of value-added carotenoids and lipids.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13068-023-02352-w.