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In addition to being important primary productive forces in marine ecosystems, diatoms are also rich in bioactive substances such as triacylglycerol and fucoxanthin. However, little is known about the transcriptional mechanisms underlying the biosynthesis of these substances. In this study, we found that the heat shock transcription factor PtHSF1 positively regulated the synthesis of triacylglycerol and fucoxanthin in Phaeodactylum tricornutum. Overexpression of PtHSF1 could increase the contents of triacylglycerol and fucoxanthin and upregulate key enzyme genes involved in the triacylglycerol and fucoxanthin biosynthesis pathways. On the other hand, gene silencing of PtHSF1 reduced the contents of triacylglycerol and fucoxanthin and the expression of the key enzyme genes involved in the triacylglycerol and fucoxanthin biosynthesis pathways. Further biochemical analysis revealed that PtHSF1 upregulated glycerol-2-phosphate acyltransferase 3 (GPAT3) and 1-deoxy- d -xylulose 5-phosphate synthase (DXS) by directly binding to their promoters, while genetic analysis demonstrated that PtHSF1 acted upstream of GPAT3 and DXS to regulate triacylglycerol and fucoxanthin synthesis. Therefore, in addition to elucidating the regulation mechanisms underlying PtHSF1-mediated triacylglycerol and fucoxanthin synthesis, this study also provided a candidate target for metabolic engineering of triacylglycerol and fucoxanthin in P. tricornutum. [ABSTRACT FROM AUTHOR]