We　investigated　the　expression　and　function　of　miR-409-5p　in　human　breast　cancer.　Quantitative　real-time　polymerase　chain　reaction　was　conducted　to　evaluate　endogenous　miR-409-5p　expression　in　breast　cancer　tumors　and　breast　cancer　cell　lines.　Lentiviral　transduction　was　performed　to　stably　downregulate　miR-409-5p　in　breast　cancer　cell　lines　MDA-MB-231　and　MCF-7　and　cells.　The　effects　of　miR-409-5p　downregulation　on　breast　cancer　proliferation,　migration,　and　xenograft　development　were　then　evaluated.　Downstream　target　gene　of　miR-409-5p,　Ras　suppressor　protein　1,　was　examined　by　dual-luciferase　activity　assay,　quantitative　real-time　polymerase　chain　reaction,　and　western　blot　in　lentiviral-transduced　breast　cancer　cells.　Ras　suppressor　protein　1　was　also　inhibited　in　miR-409-5p-downregulated　breast　cancer　cells　to　examine　its　functional　effect　on　breast　cancer　proliferation　and　migration.　MiR-409-5p　was　aberrantly　upregulated　in　both　breast　cancer　tumors　and　cell　lines.　Lentiviral　transduction　successfully　downregulated　endogenous　miR-409-5p　expression　as　well　as　suppressed　proliferation,　migration,　and　xenograft　development　in　MDA-MB-231　and　MCF-7　cells.　Ras　suppressor　protein　1　was　confirmed　to　be　directly　targeted　by　miR-409-5p　in　breast　cancer　cells.　Small　interfering　RNA-mediated　Ras　suppressor　protein　1　inhibition　reversely　promoted　cancer　proliferation　and　migration　in　miR-409-5p-downregualted　breast　cancer　cells.　MiR-409-5p　is　downregulated　in　breast　cancer　and　its　inhibition　has　anti-cancer　effect　on　breast　cancer　development　both　in　vitro　and　in　vivo.　The　regulatory　effect　of　miR-409-5p　inhibition　is　likely　through　the　inverse　upregulation　of　Ras　suppressor　protein　1　in　breast　cancer.