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Microalgae are the largest CO 2 fixer and O 2 producer on the earth and occupy an increasingly important position in human life and production. Various environmental factors have a significant impact on the growth and metabolism of microalgae. As global warming intensifies, heat stress has become a crucial factor affecting the microalgae industry. However, till now, it has not been clear how microalgae sensed the temperature stress, transmitted stress signals and adjusted in intracellular metabolic pathways. In this study, the growth of microalgae Auxenochlorella protothecoides UTEX2341 was inhibited at 32 °C, but the single cell dry weight increased. The cell component analyses showed that both the carbohydrate and total protein content decreased significantly, while the lipid content increased by 158%. Meanwhile, the intracellular Ca 2+ concentration increased continuously, with a maximum increase of 1.65 times. According to the transcriptome analyses, the up-regulation of Ca 2+ influx channel protein mid1-complementing activity 1 (MCA1) gene and the down-regulation of efflux channel protein cation exchanger 1(CAX) and autoinhibited Ca 2+ -ATPase 1 (ACA1) genes in cytoplasmic membrane jointly facilitated the increase of Ca 2+ in the cytoplasm. Coexpression network analysis indicated that the fluctuation of Ca 2+ in the cytoplasm could activate the expression of transcription factors MYB3 and AP2-4 through calmodulin (CAM) and calcium-dependent protein kinase (CDPK), and then regulate glycerol-3-phosphate acyltransferases (GPAT) at the beginning of TAG synthesis and diacylglycerol acyltransferase (DGAT)/phospholipid: diacylglycerol acyltransferase (PDAT) in the last step of TAG synthesis. Furthermore, the addition of Ca 2+ specific chelator BAPTA-AM inhibited the expression of GPAT, which was consistent with the decrease in microalgae lipid content. The results proved that Ca 2+ participated in the regulation of microalgae TAG synthesis under heat stress, which provided a new view for the understanding of the microalgae lipid accumulation mechanism., (Copyright © 2022 Elsevier Inc. All rights reserved.)