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As a tropical flower, <i>Nymphaea lotus</i> is a typical night-blooming waterlily used in water gardening. Its petals are rich in aromatic substances that can be used to extract essential oils and as flower tea. However, the short life of the flower seriously affects the development of its cut flowers. At present, neither the mechanism behind the night-opening waterlily flower’s opening and closing nor the difference between day-opening and night-opening waterlily flowers’ opening and closing mechanisms are clear. In this study, endogenous hormone contents of closed (CP) and open (OP) petals were measured, and transcriptome analysis of CP and OP petals was carried out to determine the signal transduction pathway and metabolic pathway that affect flower opening and closing. ABA and cell wall modification were selected as the most significant factors regulating flowering. We used qRT-PCR to identify the genes involved in the regulation of flower opening in waterlilies. Finally, by comparing the related pathways with those of the diurnal type, the obvious difference between them was found to be their hormonal regulation pathways. In conclusion, the endogenous ABA hormone may interact with the cell wall modification pathway to induce the flowering of <i>N. lotus</i>. Our data provide a new direction for the discovery of key factors regulating the flower opening and closing of <i>N. lotus</i> and provide basic theoretical guidance for future horticultural applications.