Zinc oxide (ZnO) nanoparticles (NPs) are soluble in water and can release Zn2+, an essential mineral that promotes the growth of plant cells. When ZnO NPs were administered to tobacco (Nicotiana tabacum cv. Samusun-NN) callus under white light irradiation, a concentration-dependent increase in weight was observed. Specifically, an increase in chlorophyll levels was triggered by blue light and induced by ZnO NPs. mRNA-seq analysis showed that during the early stages of tobacco callus exposure to ZnO NPs and white light irradiation, there was considerable fluctuation in the expression of genes related to salt stress. After 24 h, the expression of cellular component and growth-related genes also fluctuated. Analysis by RT-qPCR revealed that, after 1 day of ZnO NPs exposure, the expression levels of photosynthesis-related genes were enhanced. The Zn content of control-treated callus was 0.19 mg g−1 dry weight, whereas that of callus cultured with the ZnO bulk particles (BPs), with a particle diameter of 2000 nm, was 2.59 mg g−1 dry weight, and for callus cultured with ZnO NPs, with a particle diameter 34 nm, the Zn content was 3.37 mg g−1 dry weight. These results indicate that ZnO particles supplied large amounts of Zn to the callus, suggesting that the smaller the particle size, the larger the surface area of particles dissolve zinc ions more efficiently and the more ions are supplied to tobacco callus cells, and resulting in an increase in plant productivity. Under the light illumination, incubation of tobacco callus with zinc oxide nanoparticle dispersion resulted in supply of much zinc ions into cell, and induction of chlorophyll accumulation and cell proliferation.