Objective To establish a human bladder cancer cell line that stably overexpresses miR-449c. Methods The DNA fragments encoding pre-miR-449c were amplified from genomic DNA templates of HEK293 cells using high-fidelity PCR and cloned into FtetUGW-T vector. The recombinant plasmid was verified by sequencing and applied for lentivirus packaging. Human bladder cancer 5637 cells were infected with the lentivirus and the infected cells after selection with puromycin were observed using an inverted fluorescent microscope for enhanced green fluorescent protein (EGFP) expression, real-time quantitative PCR for miR-449c expression and Western blotting for the expression of its downstream target C-myc. Results The FtetUGW-T/miR-449C lentivirus was successfully established. The human 5637 bladder cancer cells infected with the lentivirus expressed EGFP and overexpressed miR-449c. The expression of c-myc in the cells that stably overexpressed miR-449c was significantly reduced compared with control cells. Conclusion 5637 bladder cancer cell line that stably overexpresses miR-449c is successfully established.