In this study, the tobacco NtSKOR1 gene that is homologous to AtSKOR gene in Arabidopsis was identified from tobacco via NCBI Blast and RTPCR. The bioinformatics and expression characteristics of NtSKOR1 were analyzed, and the knock out materials of NtSKOR1 was obtained by CRISPR/Cas9 technology. The results showed that: (1) full length CDS of NtSKOR1 was found to be 2 466 bp, encoding a sequence of 821 amino acid residues, and successfully cloned with gene specific primer pair by RTPCR. The isoelectric point of this protein is 6.36 and the molecular weight is 94.21 kD. (2) The subcellular localization result of NtSKOR1 showed that this gene was localized in cell membrane. Amino acid sequence analysis showed that this protein contains 6 transmembrane structures, but with no signal peptide sequence. As a typical SKOR family protein, NtSKOR1 is featured by poreforming domain and ankyrin structural domains. (3) Phylogenetic analysis showed that NtSKOR1 protein is close to SKOR proteins identified from tomato and potato, while is distant from the SKOR proteins of Gramineae species. (4) Tissue specific expression analysis showed that NtSKOR1 was preferentially expressed in roots, which was in line with AtSKOR1 expression pattern in Arabidopsis. After potassium stress treatment, the expression pattern of NtSKOR1 showed a fluctuant trend. (5) CRISPR/Cas9based genome editing of NtSKOR1 significantly decreased the potassium content in the leave of transgenic lines, suggesting NtSKOR1 gene is one of the key genes in controlling potassium content in tobacco leaves. Our results provided significant evidence of the molecular mechanism of potassium intake and transport in tobacco.