BACKGROUND Chemoresistance is a main limitation in chemotherapy for therapeutic cancer. MicroRNA (miRNA) has been indicated in the progression and tumorigenesis of many types of cancer, but the effect of miR-34b-3p in bladder cancer (BCa) cells is still unknown. MATERIAL AND METHODS This research compared the multidrug-sensitive (5637) BCa cell line and the multidrug-resistant (EJ) BCa cell line. We found that CCND2 (G1/S-specific cyclin-D2) and P2RY1 (purinergic receptor P2Y1) were the targets of miR-34b-3p, as further validated by qRT-PCR (quantitative real-time polymerase chain reaction) and western blot analysis. RESULTS Forced reversal of the levels of miR-34b-3p or CCND2/P2RY1 changed the chemoresistance profiles in both 5637 cells and EJ cells. Further experiments suggested that the CCND2 gene and the P2RY1 gene act in concert to negatively correlate with miR-34b-3p effect on BCa multidrug-chemoresistance. CONCLUSIONS These results not only reveal new players regulating BCa chemoresistance, but also provide clues for effective chemotherapy for BCa patients.