Soil salinity is expanding globally. A major concern is the toxicity of sodium ion (Na+), which results from their excessive accumulation in plant tissues and leading to plant death. The plasma membrane-localized Na+/H+ antiporter, SALT OVERLY SENSITIVE 1 (SOS1), is the only reported transporter involved in Na+ exclusion. To comprehend plant Na tolerance mechanisms, it is crucial to identify the sites of Na+ exclusion from roots mediated by SOS1. However, there are two conflicting hypotheses regarding the sites of SOS1-mediated Na+ exclusion: whether it occurs only at the root tip or throughout the entire root, encompassing both the root tip and the mature root zone. Therefore, we directly verified whether SOS1-mediated Na+ exclusion occurs in the mature root zone by conducting radiotracer experiments. To quantitatively measure Na+ exclusion separately in the root tip and the mature root zone, we devised an air gap gel system. The experiment revealed that wild-type plants (WT) excluded more Na+ from the mature root zone than SOS1 knockout mutants (sos1). In contrast, sos1 accumulated more Na+ in mature roots than WT. These results indicate that SOS1-mediated Na+ exclusion occurs in the mature root zone. We also examined Na+ recirculation, where Na+ from aboveground is transported to the roots through the phloem. Radiotracer experiments indicated that most aboveground-derived Na+ is excluded from the roots, with SOS1 playing a significant role, particularly in the mature root zone. These findings offer new insights into Na+ dynamics within plants, which are essential for addressing global agricultural salinity problems.