The anchorage-independent colony growth of cancer cells is reportedly correlated with the tumor-forming activity; however, the correlation between the morphophenotype of each colony and the tumor-forming activity has not been clarified. To assess this problem, we cultured single A549 cells (human lung adenocarcinoma cell line) in growth medium in individual wells (n = 426) for 14 days under anchorage-independent conditions and analyzed the resulting growth characteristics. The single A549 cells formed various sizes of floating colonies. The proportion of large colonies (>400 μm) was 3.8% and this proportion increased dramatically with the exogenous addition of EGF (21.6%) or HGF (27.6%). Morphologically, the floating colonies could be divided into: (ii) Type A, spheroid colony; and (ii) Type B, dispersed villous colony. The Type B colonies expressed significantly higher levels of epithelial-mesenchymal transition (EMT)-related mRNAs (Snail 1, ZEB 1, and ZEB2) than the Type A colonies. Furthermore, the subcutaneous injection of a single cell-derived colony with a large size and a Type B morphology resulted in more efficient tumor formation. The present results indicated that the morphophenotypes of floating colonies derived from single cancer cells have a critical impact on tumor-forming activity.