Respiratory syncytial virus (RSV) is the most common respiratory pathogen in infants and young children. The pathophysiology of this infection in the respiratory system has been studied extensively, but little is known about its consequences in other systems. We studied whether RSV infects human bone marrow stromal cells (BMSCs) in vitro and in vivo, and investigated whether and how this infection affects BMSC structure and hematopoietic support function. Primary human BMSCs were infected in vitro with recombinant RSV expressing green fluorescent protein. In addition, RNA from naive BMSCs was amplified by PCR, and the products were sequenced to confirm homology with the RSV genome. The BMSC cytoskeleton was visualized by immunostaining for actin. Finally, we analyzed infected BMSCs for the expression of multiple cytokines and chemokines, evaluated their hematopoietic support capacity, and measured their chemotactic activity for both lymphoid and myeloid cells. We found that BMSCs support RSV replication in vitro with efficiency that varies among cell lines derived from different donors; furthermore, RNA sequences homologous to the RSV genome were found in naive primary human BMSCs. RSV infection disrupted cytoskeletal actin microfilaments, altered cytokine/chemokine expression patterns, decreased the ability of BMSCs to support B cell maturation, and modulated local chemotaxis. Our data indicate that RSV infects human BMSCs in vitro, and this infection has important structural and functional consequences that might affect hematopoietic and immune functions. Furthermore, we have amplified viral RNA from naive primary BMSCs, suggesting that in vivo these cells provide RSV with an extrapulmonary target.