Partial root-zone irrigation (PRI), a water-saving technique, improves water uptake in hydrated roots by inducing specific responses that are thought to be regulated by signals originating from leaves; however, this signaling is poorly understood. Using a split-root system and polyethylene glycol 6000 to simulate PRI in cotton (Gossypium hirsutum), we showed that increased root hydraulic conductance (L) and water uptake in the hydrated roots may be due to the elevated expression of cotton plasma membrane intrinsic protein (PIP) genes. Jasmonate (jasmonic acid [JA] and jasmonic acid-isoleucine conjugate [JA-Ile]) content and the expression of three JA biosynthesis genes increased in the leaves of the PRI plants compared with those of the polyethylene glycol-free control. JA/JA-Ile content also increased in the hydrated roots, although the expression of the three JA genes was unaltered, compared with the control. The JA/JA-Ile contents in leaves increased after the foliar application of exogenous JA and was followed by an increase in both JA/JA-Ile content and L in the hydrated roots, whereas the silencing of the three JA genes had the opposite effect in the leaves. Ring-barking the hydrated hypocotyls increased the JA/JA-Ile content in the leaves but decreased the JA/JA-Ile content and L in the hydrated roots. These results suggested that the increased JA/JA-Ile in the hydrated roots was mostly transported from the leaves through the phloem, thus increasing L by increasing the expression of GhPIP in the hydrated roots under PRI. We believe that leaf-derived JA/JA-Ile, as a long-distance signal, positively mediates water uptake from the hydrated roots of cotton under PRI.