microRNA (miRNA) regulate protein expression levels either by degrading mRNA or repressing translation. Circulating miRNAs have been proposed as biomarkers for different diseases. One particular miRNA i.e. miR-223, is abundant in platelets and markedly downregulated in plasma from patients with diabetes or at a high risk of myocardial infarction. The aim of this study was to determine whether miR-223 is only a biomarker or whether changes in miR-223 also affect platelet function. Two models of miR-223 deletion in platelets were used; miR-223 knockout mice (miR-223 -/-) and platelets from diabetic patients. Values are means ± S.E.M., compared either by ANOVA or the Student t test.Platelets from miR-223-/- mice demonstrated an enhanced platelet aggregation in response to either thrombin (n=8) or collagen (n=22) compared to platelets from wild-type littermates (C57BL/6). Moreover, platelets from miR-223-/- mice developed large aggregates when added to different extracellular matrices such as collagen, fibronectin, and laminin and demonstrated a delayed clot retraction. Platelets from diabetic patients also formed large aggregates (339.8 µm2 ± 60 for diabetic patients vs. 78.46 µm2 ± 40.4 for healthy donors, p<0.05) and showed delayed clot retraction when compared to healthy donors. In miR-223-/- mice, the bleeding time was markedly shorter (419.7 sec ± 67.36 miR-223-/-, vs. 1177 sec ± 101.3 wild-type, p<0.001) and a significantly larger thrombus was developed after FeCl3-induced carotid artery injury than in wild-type mice (p<0.001, n=6). Furthermore, FACS analysis of whole blood showed an enhanced platelet-leukocyte aggregate (0.13 % ± 0.01 miR-223-/- , vs. 0.07% ± 0.008 wild-type, p<0.01) in miR-223-/- mice compared to wild-type. To identify proteins regulated by miR-223 we compared the proteome of miR-223-/- and wild-type platelets using 2D-differential in-gel electrophoresis. Validation of proteins identified by the proteomic analysis confirmed that miR-223-/- platelets expressed higher levels of kindlin-3, β1 integrin, α2 integrin and factor XIII-A (n=13) than wild-type platelets. The same changes in protein expression were also detected in platelets from diabetic patients (n=5). The α2β1 integrin antagonist; rhodocetin (5µg/ml, 5 min), prevented the changes in aggregate formation of platelets from miR-223-/- and diabetic patients. Additionally, factor XIII-A inhibition normalized the clot retraction process. In conclusion, our data indicate that miR-223 plays an important role in the regulation of platelet function through targeting integrin signaling and factor XIII-A expression and activity.