Primary bovine retinal microvascular endothelial cells (RMECs) are widely used for angiogenesis-based studies. Ca2+ signalling is a fundamental part of many cellular functions including the responses of RMECs to angiogenic stimuli and this relies upon appropriate regulation of gene expression. MicroRNAs (miRNA) are endogenous, non-coding RNAs which regulate mRNA expression at the post-transcriptional level. The ability of miRNAs to target multiple genes suggests that they can potentially modulate the expression of several genes in a specific pathway. We therefore investigated the possibility that miRNAs can regulate Ca2+ responses in RMECs by targeting genes of the IP3- Ca2+ signalling pathway. Both mRNA and miRNA expression in RMECs was quantified by high throughput, next generation sequencing (NGS) on an Illumina Genome Analyzer. Predicted target genes of the miRNAs expressed in RMECs were downloaded from the miRNA prediction algorithm TargetScan (1) and were compared with those genes in the Ca2+ signalling pathway which were shown to be expressed in RMECs. MiRNA-26a (miR-26a) was selected for further analysis as it was found to be within the top 20 most highly expressed miRNAs in RMECs and it was predicted to target key genes of the IP3- Ca2+ signalling pathway found to be expressed in these cells (IP3 receptor type 1 (ITPR1) and phospholipase C beta 1 (PLCB1). Overexpression (oe) of miR-26a from a plasmid vector, pSM30 (2) or knockdown (kd) with an LNA inhibitor, followed by RT-PCR, Western blotting (WB) and luciferase assays confirmed ITPR1 as a target at the protein level (3.5 fold decrease upon miR-26a oe vs. scrambled oe control and a 2 fold increase in miR-26a kd vs. scrambled kd control for WB, n=5, p<0.05, Student’s paired t-test). Uridine triphosphate (UTP) was found to induce a transient increase in RMEC [Ca2+]i that was dependent on IP3-Ca2+ signalling, as determined by the use of known IP3 receptor inhibitors, xestospongin C (3) or 2-aminoethoxydiphenyl borate (2APB) (4) and fura-2 Ca2+ microfluorimetry. Overexpression of miR-26a decreased the elevation in [Ca2+]i observed in response to UTP and knockdown of miR-26a increased the response (0.49±0.02 change in fura-2 340/380 nm ratio in scrambled oe control decreased to 0.13±0.02 upon miR-26a oe, and increased from 0.44±0.02 in scrambled kd control to 0.69±0.04 upon miR-26a kd. Mean data ± SEM, n=12, p<0.001, one-way ANOVA). Taken together, our results suggest that miR-26a, can modulate the Ca2+ responses of RMECs by targeting the IP3-Ca2+ signalling pathway. Further studies are now warranted to determine the functional significance of miR-26a in both retinal microvascular endothelial cell physiology and pathophysiology.