MicroRNAs (miRNAs) are emerging as critical regulators in carcinogenesis and tumor progression. Recently, miR-486-5p has been proved to play an important role in several cancers, but its functions in the context of breast cancer (BC) remain unknown. In this study, we found that miR-486-5p expression is significantly downregulated in BC tissues and cell lines. Overexpression of miR-486-5p dramatically suppressed BC cell proliferation in vitro and in vivo, induced G0/G1 arrest, and promoted apoptosis. We subsequently identified the oncogene PIM-1 as a direct target of miR-486-5p in BC. Overexpression of PIM-1 attenuated the function of miR-486-5p in BC cells. Together, we conclude that miR-486-5p exerts its antiproliferative function by directly downregulating PIM-1 expression. This novel miR-486-5p/PIM-1 axis provides insight into the pathogenesis of BC and might be therapeutic targets for prevention or treatment of BC.