The multicellular spheroid model partly mimics tumor microenvironments in vivo and has been reported in plenty of studies regarding radiosensitivity. However, clear isolation of quiescent and proliferating cells in live conditions has been quite difficult owing to technical limitations; therefore, comprehensive characterization could not be done thus far. In this study, we succeeded in separately isolating different cell types using a fluorescent ubiquitination-based cell cycle indicator (Fucci) and determining their radiosensitivities. Unexpectedly, proliferating cells were more radioresistant than quiescent cells due to the contact effect when spheroids were disaggregated immediately after irradiation. However, the radiosensitivity of quiescent cells was not influenced by mild hypoxia (hypoxia-inducible factor-1α-positive but pimonidazole-negative), but their radioresistance became similar to that of proliferating cells due to potentially lethal damage repair when disaggregated 24 h after irradiation. The Fucci system further allowed long-term observation of cell kinetics inside of the spheroid following irradiation using real-time confocal fluorescence scanning. Repeated cycles of recruitment from the quiescent to the proliferating phase resulted in cell loss from the outside of the spheroid toward the inside, causing gradual shrinkage. Interestingly, the central region of the spheroid entered a dormant stage approximately 40 days after irradiation and survived for more than 2 months. Using the Fucci system, we were able to comprehensively characterize the radiosensitivity of spheroids for the first time, which highlights the importance of cell cycle kinetics after irradiation in determining the radiosensitivity under tumor microenvironments.