Abstract: The Stelar K⁺ outward rectifier channel (SKOR) is a type of outwardly-rectifying Shaker K⁺ channel located in the plasma membrane of column cells in plant roots. This study aimed to investigate the promoter activity and expression of the NtSKOR1 gene at different developmental stages. For this purpose, a 2439 bp promoter segment upstream of the start codon of NtSKOR1 was cloned and ProNtSKOR1::GUS transgenic lines were generated. GUS staining and RT-qPCR assays were conducted to assess the activity and expression, respectively. The prediction of cis-acting elements revealed the presence of several elements related to light, stress, and hormone responses. During the full seedling stages, no evident GUS activity was observed. At the small cross-shaped stage, GUS activity was detected in the stem apex meristem tissues and the veins of two true leaves. During the root spreading stage, GUS activity was mainly observed in the vascular tissues of the stem, and weak activity began to appear in the vascular tissues of root and gradually increased as the tobacco plants grow. At the most flowering stage, the activity was primarily detected in the vascular tissues of the roots, stems, and upper leaves of tobacco. These findings were consistent with the results of RT-qPCR analysis. In conclusion, the expression of NtSKOR1 initiates at the small cross-shaped stage in tobacco plants and is mainly localized in the vascular tissues of the roots, stems, and leaves. Furthermore, at the most flowering stage, NtSKOR1 expression is significantly higher in the upper leaves, which have strong photosynthetic capacity, compared to the lower leaves with weak photosynthetic capacity. This suggests that NtSKOR1 may play a crucial role in the transport of K⁺ ions and photosynthetic products in tobacco plants.