Extracellular freezing results in cellular dehydration caused by water efflux, which is likely regulated by aquaporins (AQPs). In a seasonal cold acclimation (CA) study of Rhododendron catawbiense, two AQP cDNAs, RcPIP2;1 and RcPIP2;2, were down-regulated as the leaf freezing tolerance (FT) increased from −7 to ∼−50 °C. We hypothesized this down-regulation to be an adaptive component of CA process allowing cells to resist freeze-induced dehydration. Here, we characterize full-length cDNAs of the two Rhododendron PIPs, and demonstrate that RcPIP2s have water channel activity. Moreover, RcPIP2s were over-expressed in Arabidopsis, and FT of transgenic plants was compared with that of wild-type (WT) controls. Data indicated a significantly lower constitutive FT and CA ability of RcPIP2-OXP plants (compared with WT) due, presumably, to their lower ability to resist freeze desiccation. A relatively higher dehydration rate of RcPIP2-OXP leaves (than WT) supports this notion. Phenotypic and microscopic observations revealed bigger leaf size and mesophyll cells of RcPIP2-OXP plants than WT. It is proposed that lower FT of transgenic plants may be associated with their leaf cells' propensity to greater mechanical stress, that is, volume strain per unit surface, during freeze–thaw-induced contraction or expansion. Additionally, greater freeze injury in RcPIP2-OXP plants could also be attributed to their susceptibility to potentially faster rehydration (than WT) during a thaw.