Nanowires are presently investigated in the context of various biological and medical applications. In general, these studies are population-based, which results in sub-populations being overlooked. Here, we present a single cell analysis of cell cycle and cell movement parameters of cells seeded on nanowires using digital holographic microscopy for time-lapse imaging. MCF10A normal-like human breast epithelial cells and JIMT-1 breast cancer cells were seeded on glass, flat gallium phosphide (GaP), and on vertical GaP nanowire arrays. The cells were monitored individually using digital holographic microscopy for 48 h. The data show that cell division is affected in cells seeded on flat GaP and nanowires compared to glass, with much fewer cells dividing on the former two substrates compared to the latter. However, MCF10 cells that are dividing on glass and flat GaP substrates have similar cell cycle time, suggesting that distinct cell subpopulations are affected differently by the substrates. Altogether, the data highlight the importance of performing single cell analysis to increase our understanding of the versatility of cell behavior on different substrates, which is relevant in the design of nanowire applications.