Cold acclimation process plays a vital role in the survival of chilling- and freezing-tolerant plants subjected to cold temperature stress. However, it remains elusive whether a cold acclimation process enhances root water uptake (a component of chilling tolerance) in chilling-sensitive crops such as rice. By analyzing the root hydraulic conductivity under cold stress for a prolonged time, we found that cold stress induced a gradual increase in root osmotic hydraulic conductivity [Lp(r(os))]. Compared with the control treatment (roots and shoots at 25°C), low root temperature (LRT) treatment (roots at 10°C; shoots at 25°C) dramatically reduced Lp(r(os)) within 1 h. However, Lp(r(os)) gradually increased during prolonged LRT treatment and it reached 10-fold higher values at day 5. Moreover, a coordinated up-regulation of root aquaporin gene expression, particularly OsPIP2;5, was observed during LRT treatment. Further, comparison of aquaporin gene expression under root-only chilling (LRT) and whole-plant chilling conditions, and in the roots of intact plants vs. shootless plants, suggests that a shoot to root signal is necessary for inducing the expression of aquaporin genes in the root. Collectively, these results demonstrate that a cold acclimation process for root water uptake functions in rice and is possibly regulated through aquaporins.