Invasive cancer cells are a critical target in order to prevent metastasis. In the present report, we demonstrate real-time visualization of cell cycle kinetics of invading cancer cells in 3-dimensional (3D) Gelfoam (R) histoculture, which is in vivo-like. A fluorescence ubiquitination cell cycle indicator (FUCCI) whereby G(0)/G(1) cells express a red fluorescent protein and S/G(2)/M cells express a green fluorescent protein was used to determine the cell cycle position of invading and non-invading cells. With FUCCI 3D confocal imaging, we observed that cancer cells in G(0)/G(1) phase in Gelfoam (R) histoculture migrated more rapidly and further than cancer cells in S/G(2)/M phases. Cancer cells ceased migrating when they entered S/G(2)/M phases and restarted migrating after cell division when the cells re-entered G(0)/G(1). Migrating cancer cells also were resistant to cytotoxic chemotherapy, since they were preponderantly in G(0)/G(1), where cytotoxic chemotherapy is not effective. The results of the present report suggest that novel therapy targeting G(0)/G(1) cancer cells should be developed to prevent metastasis.