The circadian clock regulates plant tissue hydraulics to synchronize water supply with environmental cycles and thereby optimize growth. The circadian fluctuations in aquaporin transcript abundance suggest that aquaporin water channels play a role in these processes. Here, we show that hydraulic conductivity (Kros) of Arabidopsis (Arabidopsis thaliana) rosettes displays a genuine circadian rhythmicity with a peak around midday. Combined immunological and proteomic approaches revealed that phosphorylation at two C-terminal sites (Ser280, Ser283) of PLASMA MEMBRANE INTRINSIC PROTEIN 2;1 (AtPIP2;1), a major plasma membrane aquaporin in rosettes, shows circadian oscillations and is correlated with Kros. Transgenic expression of phosphodeficient and phosphomimetic forms of this aquaporin indicated that AtPIP2;1 phosphorylation is necessary but not sufficient for Kros regulation. We investigated the supporting role of 14-3-3 proteins, which are known to interact with and regulate phosphorylated proteins. Individual knockout plants for five 14-3-3 protein isoforms expressed in rosettes lacked circadian activation of Kros. Two of these [GRF4 (14-3-3Phi); GRF10 (14-3-3Epsilon)] showed direct interactions with AtPIP2;1 in the plant and upon coexpression in Xenopus laevis oocytes and activated AtPIP2;1, preferentially when the latter was phosphorylated at its two C-terminal sites. We propose that this regulatory mechanism assists in the activation of phosphorylated AtPIP2;1 during circadian regulation of Kros.