Carbon nanotubes concentration modulates endogenous hormone balance, influencing callogenesis and organogenesis efficiency, with potential for optimizing plant transformation programs. A unique feature of plant somatic cells is their remarkable ability to regenerate new organs and even an entire plant in vitro. In this work, we investigated how an important group of environmental factors, carbon nanotubes (CNTs) (both single-walled nanotubes as SWCNTs and multi-walled nanotubes as MWCNTs), affect the regenerative capacity of plants and the underlying molecular mechanisms. Our data show that both the induction of pluripotent callus from Arabidopsis root explants and the frequency of de novo shoot regeneration were influenced by the concentration, but not the type of CNTs. Raman analyses show that CNTs can be transported and accumulate in the callus tissue and in the newly formed seedlings. The contrasting effects of CNTs at 0.1 mg L and 50 mg L were reflected not only in the concentrations of endogenous auxin and trans-zeatin (tZT), but also in the changes in the expression levels of positive cell cycle regulators and transcriptional regulators that control callus pluripotency and the establishment of shoot apical meristem (SAM). Since most existing plant transformation strategies involve the conversion of dedifferentiated calli into regenerated plantlets and are very time consuming and inefficient, this work suggests that CNTs could be used as an additive to optimize plant micropropagation and genetic engineering systems by modulating hormone balance and stimulating the intrinsic totipotency of plants, thus overcoming organogenic recalcitrance.