MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and play important roles in cancer development and progression. MiR-125b-5p is a miRNA that has been reported to have diverse and context-dependent effects on different cancer types and subtypes. In this study, we aimed to investigate the expression and function of miR-125b-5p in MCF-7 breast cancer cells and to explore the potential of using chitosan nanoparticles for miR-125b-5p delivery. We found that miR-125b-5p was downregulated in MCF-7 cells compared to normal mammary epithelial cells, and that its overexpression reduced the viability of MCF-7 cells by targeting Raf-1 and BMPR1b genes, which are involved in cell survival and proliferation. We also synthesized and characterized chitosan/miR-125b nanoparticles (CNPs) and evaluated their in vitro release profile and cellular uptake. We showed that CNPs enhanced the delivery and efficiency of miR-125b-5p, resulting in a more potent inhibition of Raf-1 and BMPR1b gene expression and a greater reduction of cell viability. Our results suggest that miR-125b-5p and CNPs have potential anti-tumor effects on human breast cancer cells by suppressing Raf-1 and BMPR1b gene expression. Our study provides a new insight into the role and mechanism of miR-125b-5p and its target genes in breast cancer, and demonstrates the feasibility and efficacy of using chitosan nanoparticles for miR-125b-5p delivery.