Abstract: To broaden the source of tobacco sterility genes, based on the capsicum male sterility gene Cams1 sequence, the gene Ntms1 was homologously cloned in tobacco variety K326. Bioinformatics method was employed to analyze the nucleotide sequence, protein structure, hydrophilicity and domain of Capsicum gene Cams1 and tobacco gene Ntms1, and the function of Ntms1 was verified by using CRISPR/Cas9 technique. Results showed that the protein sequence similarity of Cams1 and Ntms1 reached 85.25%, with the products encoded by the two genes having same PHD functional domain, same phosphorylation recognition site, similar secondary and tertiary protein structures, and the two genes had no signal peptide and transmembrane structure. It was speculated that tobacco Ntms1 gene and pepper sterile gene Cams1 had similar function of fertility control. The results of genetic transformation showed that the expression of Ntms1 and the amount of active pollen in the mutant were highly significantly lower than that in the wild type, which proved that Ntms1 had the function of tobacco pollen fertility control.