Individuals with Type 2 diabetes mellitus (T2DM) are at increased risk of premature atherosclerosis and coronary heart disease, and suffer poorer outcomes following surgical intervention. Smooth muscle cells (SMC) within the vessel wall play key roles in adaptation to physiological and pathological stimuli through inherent plasticity that permits phenotypic switching between quiescent, contractile and active, synthetic states. MicroRNAs (miRs) are short, non-coding RNAs that negatively regulate gene expression in a variety of pathologies and whilst roles for miRs have been associated with development of T2DM, their influence on macrovascular complications and specifically at the level of vessel wall SMC is unknown. SMC were cultured from saphenous vein explants of non-diabetic (ND) patients and those with established T2DM. Cell morphology was analysed using phase contrast microscopy and ImageJ software. Cytoskeletal structure (F-actin) was examined using rhodamine phalloidin fluorescence microscopy and cell proliferation was determined by direct cell counting. Expression of miRs was quantified by TaqMan real-time RT-PCR assays. Over-expression and knock-down of miRs was achieved by transfection of premiRs and antimiRs respectively. SMC from T2DM individuals were significantly larger than ND SMC (9181 ± 926 µm2 vs. 5621 ± 431 µm2, n=15, p<0.001) with a disorganised cytoskeleton and impaired proliferation (33% reduction, n=6, p<0.01). TaqMan assays revealed a specific 1.6-fold up-regulation of miR-143 and miR-145 in SMC from multiple T2DM patients relative to ND SMC (n=10, p<0.05). Over-expression of miR-143/145 in ND cells induced an increase in spread cell area (7641 ± 863 µm2 vs 5634 ± 319 µm2 in controls, n=4, p<0.001), truncated F-actin fibres and reduced proliferation rate (35% reduction, n=6, p<0.05), similar to that observed in native T2DM SMC. Conversely, antimiR-143/145 transfection in T2DM cells reversed the phenotype towards that of native ND SMC. In summary, SMC from T2DM patients exhibit a distinct phenotype that exhibits features of both contractile and synthetic cells. Importantly, these characteristics are maintained throughout culture and passaging, suggestive of metabolic memory. Our data support the hypothesis that aberrant expression of miR-143/145 in vascular SMC confers a persistent “T2DM phenotype”. The perceived detrimental effects of miR-143/145 may impact on the functional ability of SMC to remodel the vessel wall and contribute to vascular complications in patients with T2DM. Thus, miR-143/145 may be attractive targets for therapeutic intervention.