Flowers of Eustoma grandiflorum open in the morning and close in the evening, showing diurnal rhythms. In this study, the process of flower opening and closure of E. grandiflorum ‘Azuma-no-Murasaki’ was examined under different light cycles by capturing corolla images using interval photography. At 24-hour light cycles, the flower opening rhythm synchronized with the light cycles, and the process was composed of dual steps. The first one was immediate opening and closure at the times of initiation and cessation of the light period, respectively. The second one was gradual opening and closure, which occurred 12 hours after the end of the former light period and 2–3 hours after the initiation of the current light period, respectively. The first response appeared to be a direct effect of light, while the second one appeared to be under the regulation of circadian clocks. Under constant dark, blue, or red conditions, flowers showed circadian oscillations of 25.5 ± 0.6, 25.6 ± 0.6, or 24.3 ± 0.4 hours, respectively. Under constant white light or co-irradiation of blue and red light, flowers opened and closed once, but the oscillations did not continue thereafter. The synchronization of flower opening and closure rhythms to 24- and 20-hour day cycles was observed for both blue light and red light cycles. The synchronization was not complete for 16-hour light cycles and the flower oscillation period became 24 hours under 12-hour light cycles. The direct effect of light was found to be dependent on light intensity. When blue light intensity was adjusted at 25, 40, or 100 W·m⁻², flowers opened more rapidly after illumination at a stronger light intensity, but such intensity-dependent effect was not observed for red light.