Low-temperature stress significantly reduces rice yield and grain quality. Identifying major cold-tolerance genes and deciphering their regulatory networks is therefore essential for rice molecular breeding for cold tolerance. Here, through a genome-wide association study (GWAS) in a multi-parent advanced generation intercross (MAGIC) population, we identified OsNHX2, a tonoplast-localized K⁺ transporter that enhances cold tolerance at the seedling stage. OsNHX2 enhances cold tolerance by promoting an increase in potassium ion content in shoots, which stimulates proline accumulation and reduces the levels of reactive oxygen species (ROS) and malondialdehyde (MDA). Natural variation in the coding region of OsNHX2 underlies differences in K⁺ transport ability. We further demonstrated that OsNHX2 expression is negatively regulated by the transcription factor OsATH1. One natural variant in the OsNHX2 promoter, a 45-bp deletion, elevates OsNHX2 expression by reducing OsATH1 binding. Notably, we found that OsNHX2 physically interacts with OsGAPC3, a cytosolic glyceraldehyde-3-phosphate dehydrogenase known to modulate responses to abiotic stresses via ROS signaling. This interaction masks the tonoplast-targeting signal of OsNHX2, altering its subcellular localization, impairing K⁺ transport, and promoting cytosolic K⁺ accumulation. Overexpression of OsNHX2 significantly increased the seed-setting rate under cold stress at the booting stage without compromising grain yield under normal conditions. Haplotype analysis revealed that rice accessions carrying the cold-tolerant OsNHX2 haplotype are predominantly distributed in higher-latitude regions, suggesting adaptive selection. Our findings demonstrate that OsNHX2, which is negatively regulated by OsATH1 and OsGAPC3 at both transcriptional and post-transcriptional levels, modulates intracellular K⁺ accumulation to control cold tolerance in rice.