The aim of this study was to find novel tumor suppressor microRNAs through screening genes epigenetically silenced by methylation in bladder cancer (BC) cell lines using microRNA microarrays. Since miR-517a and miR-520g, both located on chromosome 19q13.42, were found to highly up-regulated genes after treatment with a demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dc), we hypothesized that they are tumor-suppressor microRNAs and performed a gain-of-function study using these mature microRNAs. The miR-517a restoration showed significant inhibition of cell proliferation in the transfectants compared to miR-control-transfected cells (p<0.0001 both in BOY and T24 cells). Furthermore, ectopic overexpression of miR-517a markedly induced apoptosis in the miR-517a-transfected BC cell lines. In addition, we carried out oligo microarray analysis using miR-517a transfectants and miR-control transfectants (BOY and T24), from which 35 down-regulated genes and 19 up-regulated genes were identified. These included amphiregulin (AREG) and BCL2-associated transcription factor 1, transcript variant 1 (BCLAF1), previously reported to be concerned with apoptosis, in both cell lines by miR-517a restoration. These data suggest that miR-517a functions as a tumor suppressor through inhibition of cell proliferation and induction of apoptosis under the regulation of AREG and/or BCLAF1 in BC cells. Anti-apoptotic effects may be maintained by down-regulation of miR-517a due to DNA hypermethylation in human BC cells, suggesting that restoration of miR-517a may be a novel therapeutic strategy for human BC.