Objective: The pro-differentiation microRNA gene MIR143/145 and the pro-proliferation gene MIR221/222 play critical roles in vascular injury and vascular smooth muscle cell (SMC) phenotypic modulation. The goals of this study were to 1) identify soluble factors that regulate MIR143/5 and MIR221/2 expression, and 2) test the effects of MIR145 depletion on SMC gene expression. Methods: Rat aortic SMCs were treated for 24 hours with interleukin-1-b (IL1b), angiotensin-II (ANGII), endothelin-1 (ET1), sphingosine-1-phosphate (S1P), or oxidized phospholipids (OXPAPC). Mature microRNA levels were measured by real-time RT-PCR. MIR143/5 and SM a-actin (ACTA2) promoter-luciferase constructs were used to assay promoter activity. MIR145 was reduced by transfection of an oligo-based inhibitor. Results: The contractile agonists ANGII and ET1 did not affect MIR143/5 and 221/2 levels or MIR143/5-luc activity. IL1b increased MIR222 levels 88% compared to vehicle control. S1P increased MIR143 and 145 levels 24% and 20%, respectively, and increased MIR143/5-luc activity by 37%. OXPAPC increased MIR221 and 222 by 2.1-fold and 3.7-fold, respectively. OXPAPC had no effect on MIR143/5 levels; however, MIR143/5-luc activity was decreased 70%. OXPAPC also decreased ACTA2 (86%) and myocardin (70%) mRNA levels, and increased KLF4 mRNA 135-fold. We also show that a MIR145 inhibitor reduced basal ACTA2-luc activity 88%, ACTA2 mRNA 60%, myocardin mRNA 43%, and increased KLF4 mRNA 9.3-fold. Intriguingly, MIR145 knockdown increased the contractile gene transgelin's mRNA by 2.5-fold. Conclusion: MIR145 knockdown studies confirm MIR145's role in promoting the SMC contractile phenotype by targeting KLF4, a transcriptional repressor of ACTA2. Upregulation of MIR143/5 promoter activity by S1P supports its role in positively regulating ACTA2. OXPAPC, however, negatively regulates the MIR143/5 gene cluster and positively regulates the pro-proliferative and pro-migratory MIR221/2 gene cluster.