The invention relates to the technical field of genetic engineering, and relates to a gene for regulating and controlling the potassium content of tobacco leaves, in particular to an NtYpi1 gene capable of regulating and controlling the potassium content of the tobacco leaves and application of the NtYpi1 gene. The invention provides an NtYpi1 gene for regulating and controlling the potassium content of tobacco leaves. The nucleotide sequence of the NtYpi1 gene is shown as SEQ ID NO. 1. The invention further provides application of the NtYpi1 gene for regulating and controlling the potassium content of the tobacco leaves, after the gene is knocked out through CRISPR/cas9, the potassium content of the tobacco leaves is remarkably reduced compared with that of a control group, and it can be seen that the NtYpi1 gene participates in regulation and control of the potassium content of the tobacco leaves. The regulation and control function of the NtYpi1 gene on the potassium content of the tobacco leaves is defined, a new evidence is provided for analyzing a molecular regulation and control mechanism of the potassium ion content of the tobacco leaves, a new thought is provided for regulating and cultivating tobacco with improved potassium content through gene expression, and a new gene resource is provided for improving the potassium content of the tobacco leaves through a molecular means.