Multiple myeloma is characterized by frequent chromosomal alterations. Deletion of chr 13, especially band 13q14, is commonly observed in early stages of MM, suggesting the presence of tumor suppressor genes (TSG) within this region. We sought to functionally validate the role of the microRNAs-15a/16 cluster, centered at the deleted region, as TSGs and delineate their downstream target genes in MM. Using sponge lentiviral vectors to competitive stably inhibit mature microRNAs in vitro and in vivo, we have documented enhanced proliferative and invasive capacity of cells with stably inhibition of miR-16. Expression profiling analysis of miR-16-deficient cells identified a large number of downstream target genes. This loss-of-function system, which mimics the 13q chromosomal deletion, provides a valuable tool to investigate their function in MM pathogenesis and their potential use as therapeutic targets.