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Background: Circadian clock precisely control flowers open and close at specific times of day responsive to the environment, providing advantages in reproduction and adaption. Flowers of Iris dichotoma and I. domestica are ephemeral and show single opening and closing which present circadian rhythms. To reveal the key clock-related genes and mechanisms regulating flower opening and closing times, transcriptomes of two F2 hybrid plants with significantly divergent flower opening and closing times were sequenced at different flowering stages. Genes with different temporal expression patterns between the two individuals were screened.Results: PIF4-like (PHYTOCHROME-INTERACTING FACTOR4-like) whose homologous gene in Arabidopsis integrates external signals (light and temperature) and intrinsic signals from the clock would negatively regulate flower opening and positively regulate flower closing. Downstream the clock, genes related to auxin synthesis (YUC10 and YUC2-like), auxin efflux transport (auxin efflux carrier component 1, 7) and signaling (SAUR64-like and auxin-induced protein 22D) were specifically up-regulated in flower opening process (from flower buds to flower starting opening), suggesting that auxin is necessary for flower opening. Genes involved in osmoregulation, inward water transport and cell wall loosening were also specifically up-regulated in flower opening process, causing rapid cell expansion during flower opening. The start of flower closure is associated with the increased activities of cell wall degradation, protein degradation, sucrose synthesis, fatty acid degradation, phospholipid degradation and remobilization of mineral ions.Conclusions: This research illuminates the key clock-related regulator of flower opening and closing times, and improves the understanding in regulatory networks of flower opening and closing.