Carbon nanomaterials (CNMs) have attracted a great deal of research interest for their potential application in plants. Some types of CNMs have exhibited the ability on regulating plant growth of plants, which showed a promising future in agriculture. However, detailed mechanism of their regulation on plant growth is still not well characterized especially at the molecular level. In this study, our objective is to study the regulation mechanism of carbon nanoparticles (CNPs) on tobacco callus growth at protein level. During the incubation period, proteomic profiling of tobacco callus was investigated by using the isobaric tags for relative and absolute quantitation labelling (iTRAQ) coupled with 2D-LC MS/MS. The function of differentially expressed proteins was achieved by using a gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analysis with InterproScan, and qRT-PCR was used to support the results of the proteomic data. The results showed that the growth of tobacco callus was enhanced by CNPs, with the optimal concentration at 50 mg/L. The expression levels of 198 cellular proteins were significantly up- or down-regulated after CNPs treatment. Some differentially expressed proteins were located in mitochondria and involved in calcium-mediated signaling pathway. The proteomic profile changes provided more deeper understanding of the highly complex regulatory mechanisms in tobacco callus exposed to CNPs.