Hantaviruses infect human endothelial cells (ECs) and cause two diseases marked by vascular permeability defects: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Vascular permeability occurs in the absence of EC lysis suggesting that hantaviruses alter normal EC fluid barrier functions. Hantavirus-infected endothelial cells are hyper-responsive to vascular endothelial growth factor (VEGF) which directs changes in EC adherens junctions and induces paracellular permeability. However, VEGF-directed responses are regulated by cellular microRNAs (miRNAs). The hantavirus nucleocapsid (N) protein binds cellular and viral RNA and reportedly co-localizes with P-bodies where miRNAs mature. These findings suggest that hantaviruses may modify miRNA regulation of EC protein expression. Here we analyzed miRNAs within endothelial cells infected by the HPS-causing Andes hantavirus (ANDV). The levels of 352 human miRNAs were evaluated within ANDV-infected ECs using quantitative RT-PCR arrays. Fourteen miRNAs were upregulated >4-fold following infection by ANDV, including 6 miRNAs that are associated with changes in vascular integrity. These findings suggest that ANDV alters EC miRNAs that regulate cellular permeability. Nine miRNAs were downregulated >3-fold following ANDV infection. The level of miR-410 was decreased by 3,400-fold in ANDV infected cells, although the role of miR-410 in regulating EC functions is currently unknown. We evaluated ANDV-induced changes in miR-126, an endothelial cell-specific miRNA, which regulates vascular integrity by downregulating SPRED1 and PIK3R2 mRNAs. However, the level of miR-126 was only increased 2-fold by ANDV infection and, in contrast to miR-126 changes, SPRED1 and PIK3R2 mRNA levels increased 10- and 7-fold, respectively, in ANDV infected ECs but remained unchanged in ECs infected by a nonpathogenic hantavirus, Tula (TULV). Since SPRED1 increases EC permeability, we evaluated the effect of SPRED1 siRNAs on ANDV induced EC permeability. SiRNA knockdown of SPRED1 dramatically decreased the permeability of ANDV-infected ECs in response to VEGF suggesting that increased SPRED1 levels contribute to EC permeability during ANDV infection. These findings suggest the potential for ANDV to interfere with miR-126 directed downregulation of SPRED1, resulting in the enhanced EC permeability of ANDV infected cells. These results suggest that ANDV infection alters the function of specific endothelial cell miRNAs that contribute to EC barrier functions and paracellular permeability.