Glucocorticoid receptor (GR) acts as a ligand-dependent transcription factor after nuclear transport from the cytoplasm at the liganded state. Importins are docking proteins for karyopherin-mediated binding of substrate in a nuclear import pathway. To investigate the spatial and temporal relation between GR and importins, we analyzed the subcellular distribution of GR and importins in response to ligand in single living cells using fusion proteins labeled with different spectral variants of green fluorescent protein. Upon activation with ligand treatment, fluorescent protein-tagged (FP)-GR was translocated from the cytoplasm to the nucleus, showing a similar time course as FP-importin in the coexpressed cells with the fusion proteins. In contrast to FP-importin, the distribution of FP-importin was little changed upon ligand treatment in the coexpressed cells with FP-GR and FP-importin. Analysis using fluorescence resonance energy transfer proved that GR directly interacted with importin in the whole area of the cytoplasm upon ligand treatment, and detached importin shortly after nuclear import. However, direct interaction between GR and importin was not detected. These studies showed visual evidence of the nuclear importing of GR in association with importin in single living cells.