Aquaporin 3 is a channel that transports both water and glycerol. Aquaporin 3-deficient mice exhibit skin defects, including decreased glycerol content and impairment of water holding capacity, barrier recovery, and wound healing. Whether aquaporin 3 and its glycerol transporting capacity are involved in regulating keratinocyte function, we have previously shown that phospholipase D2 can metabolize phospholipids in the presence of glycerol to yield phosphatidylglycerol. We hypothesized that aquaporin 3 is involved in the regulation of keratinocyte function by a mechanism involving the interaction between aquaporin 3 and phospholipase D. Using sucrose gradient centrifugation, immunoprecipitation analysis, and confocal microscopy, we found that aquaporin 3 and phospholipase D2 colocalized in caveolin-rich membrane microdomains. In addition, aquaporin 3 expression was downregulated at the transcriptional level and glycerol uptake was reduced upon primary mouse keratinocytes to differentiation in response to an elevated extracellular calcium concentration or 1,25-dihydroxyvitamin D3. Our results suggest that aquaporin 3 and phospholipase D2 form a signaling module in lipid rafts, where aquaporin 3 transports glycerol to phospholipase D2 for the synthesis of phosphatidylglycerol. Phosphatidylglycerol, as a bioactive lipid, could potentially mediate the effects of the aquaporin 3-phospholipase D2 signaling module, with aquaporin 3 as a modulatory unit, in the regulation of keratinocyte function.