Presently, the molecular mechanisms underlying endothelial dysfunction in diabetes are not fully understood. TRPV4 channels have been shown to play an essential role in endothelial-dependent vasodilatation in various vascular beds. In the present study, we have characterised the molecular and functional expression of TRPV4 channels in bovine retinal endothelial cells (BRECs) following culture in both normal (5mM) and high glucose (25mM) conditions (72 h incubations). (Data are presented as mean±SEM). In cells cultured in normal glucose, the TRPV4 agonist, 4α-PDD (1μM) induced a rise in [Ca2+]i (fura-2 microfluorimetry) (0.2±0.03 R340/380, n=8) which could be blocked by the non-selective TRPV4 antagonist, ruthenium red (10μM) (0.06±0.01 R340/380, n=6) and siRNA-mediated knockdown of TRPV4 channel expression (0.01±0.002 R340/380, n=5). The 4α-PDD response was abolished in the absence of extracellular Ca2+ (0.001±0.00001 R340/380, n=6) and greatly reduced following Ca2+ store depletion using the SERCA pump inhibitor, CPA (20µM) (0.04±0.01 R340/380, n=6). This suggests that activation of TRPV4 channels in BRECs triggers both influx of extracellular Ca2+ and Ca2+ store release. We subsequently examined the effects of high glucose on TRPV4 mRNA expression in BRECs. TRPV4 transcripts as well as protein levels were appreciably lower in cells treated with high glucose. Following pre-incubation of BRECs with CPA, the Ca2+ influx component of the 4α-PDD response was attenuated in cells grown under high glucose conditions (0.02±0.005 R340/380, n=8). Experiments were also performed to examine TRPV4-mediated membrane currents using the whole-cell patch clamp technique. The TRPV4 specific agonist GSK1016790A (1μM) increased membrane current amplitude at -80mV (-5.14±3.17 to -12.55±5.09 pA/pF, n=6) and this was reversed by the TRPV4 antagonist HC-067047 (1μM) (-5.53±2.80 pA/pF, n=6). The effects of GSK1016790A were abolished after culturing cells in high glucose (-0.78±0.34 pA/pF before and -1.14±0.39 pA/pF after GSK1016790A; -0.99±0.34 pA/pF after HC-067047, n=8). Osmotic control experiments were undertaken using mannitol or L-glucose (20mM + 5mM D-glucose). The gene and protein expression and functional responses of TRPV4 channels were identical to those observed under normal glucose condition. These results show that the expression and function of TRPV4 channels is downregulated in retinal endothelial cells under hyperglycaemic conditions. Changes in TRPV4 expression may contribute to endothelial cell dysfunction during diabetes, an issue that warrants further investigation.