In order to investigate the biological function of tobacco pathogenesis-related protein PR10, NtPR10 was obtained from the tobacco G28, which is 483bp in length, encoding 160 amino acids. This NtPR10 belongs to pathogenesis related protein family containing the “Bet_v_I” and “P-loop” domain, related with nuclease activity. The bioinformatics analysis indicated NtPR10 did not contain transmembrane region, no signal peptide, and exhibited intracellular localisation features by TMHMM, SignalP and PrositeScan. The expression pattern of the gene in the resistant and susceptible cultivars was studied by RT-qPCR. The results showed that the NtPR10 gene was significantly up-regulated in the susceptible cultivar G28 in TMV treatment. While in resistant cultivar Sumsun NN, The NtPR10 was up-regulated at six hours after TMV infection, and down-regulated between 12 hours and the eighth day, and then increased gradually to the pre-infection level after 16th day. From above analysis, NtPR10 responses to TMV infection, and the expression trend is opposite in resistant and susceptible cultivars, suggesting that the NtPR10 has an important function in the process of TMV infection, that provides the foundation for further analysis of the biological function of NtPR10. It also provides a new method of tobacco disease control.