Plant spacing usually refers to distances between plants within and between rows in the field. Different spacing in crop planting would generally influence the size, plant architecture, economic productivity, etc. The present research provided a time course monitoring of stem development in tobacco with different plant spacing. The result showed that cambium activity, vascular bundle thickness, lignin, cellulose, and hemicellulose content, as well as the macronutrient deposition in the stem varied because of the different plant spacing. Furthermore, the genes (NtHB8s and NtNST3s) coding the homologs of HB8 and NST3 transcription factors, which are involved in plant secondary growth, were cloned in tobacco. In the time course, they also indicated diverse expression patterns among altered plant-spacing treatments. Their transcriptomic activities were validated, and the motifs that might bind transcription factors in their promoter regions were predicted. Promoters of NtHB8s and NtNST3s genes were rich in light-response elements; as a result, light might be the main environmental factor in plant spacing to regulate stem secondary growth.