The 14-3-3 family of proteins are multifunctional proteins that interact with many of their cellular targets in a phosphorylation-dependent manner. Here, we determined that 14-3-3 proteins interact with phosphorylated forms of the water channel aquaporin-2 (AQP2) and modulate its function. With the exception of sigma (σ), all 14-3-3 isoforms were abundantly expressed in mouse kidney and mouse kidney collecting duct cells (mpkCCD14). Long-term treatment of mpkCCD14 cells with the type 2 vasopressin receptor agonist dDAVP increased mRNA and protein levels of AQP2 alongside 14-3-3 beta (β) and zeta (ζ), whereas levels of 14-3-3 eta (η) and theta (θ) were decreased. Co-immunoprecipitation (co-IP) studies in mpkCCD14 cells uncovered an AQP2:14-3-3 interaction that was modulated by acute dDAVP treatment. Additional co-IP studies in HEK293 cells determined that AQP2 interacts selectively with 14-3-3 ζ and θ. Use of phosphatase inhibitors in mpkCCD14 cells, co-IP with phosphorylation deficient forms of AQP2 expressed in HEK293 cells, or surface plasmon resonance studies determined that the AQP2:14-3-3 interaction was modulated by phosphorylation of AQP2 at various sites in its carboxyl-terminus, with ser256 phosphorylation critical for the interactions. shRNA-mediated knockdown of 14-3-3 ζ in mpkCCD14 cells resulted in increased AQP2 ubiquitylation, decreased AQP2 protein half-life and reduced AQP2 levels. In contrast, knockdown of 14-3-3 θ resulted in increased AQP2 half-life and increased AQP2 levels. In conclusion, this study demonstrates phosphorylation-dependent interactions of AQP2 with 14-3-3 θ and ζ. These interactions play divergent roles in modulating AQP2 trafficking, phosphorylation, ubiquitylation and degradation.