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Background: N6-methyl-adenosine (m6A) is a prevalent RNA modification in many species. Abnormal m6A methylation levels can lead to RNA dysfunction and can cause diseases. Tobacco mosaic virus (TMV) is one of the most devastating viruses for agricultural plants. It has many hosts, particularly including tobacco and other members the family Solanaceae. However, it remains unclear whether the abnormal growth induced by TMV is associated with the m6A level. Methods: A rapid and accurate analytical method using ultra-high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry (UHPLC−HR − MS/MS) was developed to analyse the adenosine (A), cytidine (C), guanosine (G), uridine (U), and m6A contents in the tobacco leaf, and the m6A/G ratio was used to evaluate the m6A level. Subsequent protein sequence alignments were used to find the potential methylases and demethylases in Nicotiana tabacum (N. tabacum). Finally, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was used to analyse the gene expression levels of the potential methylases and demethylases in the N. tabacum leaf. Results: The results showed that TMV reduced the m6A level. Moreover, protein sequence alignments revealed partial homology among human ALKBH5, Arabidopsis (NP_001031793), and Nicotiana sylvestris (XP_009800010). The gene expression level of the potential demethylase XM_009801708 increased at 14 and 21 days in N. tabacum infected with TMV, whereas all of the potential methylases decreased. Conclusions: The reversible m6A modification in N. tabacum mRNA might represent a novel epigenetic mechanism involved in TMV. [ABSTRACT FROM AUTHOR]