The role of DNA methyltransferase 3B (DNMT3B) in tumorigenesis and development has been widely recognized; however, the mechanism underlying its action remains unclear. Considering its function in de novo methylation, we aimed to investigate whether DNMT3B plays its role via microRNA (miR)-34a promoter methylation in bladder cancer. We found that DNMT3B expression was low in 10 bladder cancer tissues and high in 20 bladder cancer tissues. miR-34a expression was higher in bladder cancer tissues with low expression of DNMT3B than that in bladder cancer tissues with high expression of DNMT3B. The level of miR-34a was negatively correlated with the level of DNMT3B. The methylation ratio of the miR-34a promoter was positively correlated with the level of DNMT3B and negatively correlated with the level of miR-34a. DNMT3B knockdown increased the expression of miR-34a and the transcriptional activity of the miR-34a promoter, while decreasing miR-34a promoter methylation. DNMT3B knockdown inhibited migration and invasion, while decreasing the protein levels of hepatocyte nuclear factor 4 gamma and Notch1 which are downstream targets of miR-34a. These inhibitory effects of DNMT3B were mitigated by the miR-34a inhibitor. In conclusion, DNMT3B silencing suppresses migration and invasion by epigenetically promoting miR-34a in bladder cancer.