Key message <p>The tonoplast-localized transporter TaTPK1-5D, interacting with TaCIPK23-4D , enhances low K⁺ tolerance in wheat by promoting vacuolar K⁺ efflux, providing a key genetic target for improving K⁺-use efficiency in breeding.</p> Abstract <p>Bread wheat (<i>Triticum aestivum</i> L.) serves as a staple food for more than one-third of the global population, and potassium (K⁺) is critical for wheat yield and quality. However, the molecular mechanisms underlying wheat survival under low-K⁺ conditions remain poorly understood. In this study, a phenotypic screening of 712 wheat accessions identified a low-K⁺ sensitive genotype (H735) and a low-K⁺ tolerant genotype (H467, namely Zhengmai 136). Measurements of K⁺ concentration and non-invasive micro-test technology revealed that the differential tolerance between the two genotypes was not attributable to root K⁺ uptake capacity, but rather to a higher vacuolar K⁺ efflux rate in H467 compared to H735. Through transcriptomic-assisted differential expression and co-expression network analysis, a tonoplast-localized K⁺ efflux transporter, TaTPK1-5D, was identified as a key candidate underlying differential low-K⁺ tolerance in wheat. Functional disruption of <i>TaTPK1-5D</i>^<i>H467</i>, but not <i>TaTPK2-3D</i>^<i>H467</i><i>/2-4D</i>^<i>H467</i><i>/3-5D</i>^<i>H467</i>, significantly reduced both low-K⁺ tolerance and vacuolar K⁺ efflux in H467. High <i>TaTPK1-5D</i> expression was consistently observed in several other K⁺-efficient wheat accessions. Importantly, yeast two-hybrid screening, bimolecular fluorescence complementation, and pull-down assays demonstrated that TaTPK1-5D interacted with the protein kinase TaCIPK23-4D. Functional disruption of <i>TaCIPK23-4D</i> led to dramatic sensitivity to low-K⁺ stress. These findings establish TaTPK1-5D as a major vacuolar K⁺ efflux transporter facilitating subcellular K⁺ remobilization under low-K⁺ conditions.</p>