Regulated urine concentration takes place in the renal collecting duct upon arginine vasopressin (AVP) stimulation, where subapical vesicles containing aquaporin-2 (AQP2) are inserted into the apical membrane instantly increasing water reabsorption and urine concentration. The reabsorped water exits via basolateral AQP3 and AQP4. Upon long-term stimulation with AVP or during thirst, expression levels of both AQP2 and AQP3 are increased, however, there is so far no evidence for short-term AVP regulation of AQP3 or AQP4. To facilitate the increase in trans-epithelial water transport, AQP3 may be short-term regulated via changes in protein-protein interactions, incorporation into lipid rafts and/or changes in steady state turn-over, which could result in changes in the diffusion behavior of AQP3. Thus, we measured AQP3 diffusion coefficients upon stimulation with the AVP mimic forskolin, to reveal if AQP3 could be short-term regulated by AVP. k-Space Image Correlation Spectroscopy (kICS) analysis of time-lapse image sequences of basolateral EGFP-tagged AQP3 (AQP3-EGFP) revealed that forskolin mediated elevation of cAMP increased the diffusion coefficient by 58% from 0.0147 ± 0.0082 µm²/sec (control) to 0.0232 ± 0.0085 µm²/sec (forskolin, p<0.05). Quantum dot conjugated antibody labeling also revealed a significant increase in AQP3 diffusion upon forskolin treatment by 44% (0.0104 ± 0.0040 µm²/sec (control) vs. 0.0150 ± 0.0016 µm²/sec (forskolin, p<0.05)). Immunoelectron microscopy showed no obvious difference in AQP3-EGFP expression levels or localization in the plasma membrane upon forskolin stimulation. Thus, AQP3-EGFP diffusion is altered upon increased cAMP, which may correspond to basolateral adaptations in response to the increased apical water readsorption.