Bladder cancer (BC) is a common disease of the urinary system. Puerarin is a flavonoid extracted from Pueraria lobata. However, the role of puerarin in BC remains unclear. Hence, this study aimed to investigate the effect of puerarin on BC cells. Cell viability, proliferation, and apoptosis were measured by CCK-8, BrdU assay, and flow cytometry analysis, respectively. The expressions of miR-16, apoptosis-related factors, and the main factors of the NF-κB pathway were analyzed by qRT-PCR and Western blot. In this study, we found that cell viability and proliferation were significantly reduced, cell apoptosis was enhanced, and the mRNA level of miR-16 was upregulated in puerarin-treated T24 cells. Further, silencing of miR-16 inhibited the decrease in cell viability and the increase in apoptosis. The expression of main factors involved in the NF-κB signaling pathway was downregulated in the puerarin group, while miR-16 silencing alleviated these downregulations. More importantly, puerarin deactivated the NF-κB signaling pathway via upregulation of miR-16. Also, miR-16 downregulated COX-2 expression via deactivation of the NF-κB signaling pathway. This study demonstrated that puerarin could inhibit cell proliferation, promote cell apoptosis, and deactivate NF-κB signaling pathway via upregulation of miR-16 in T24 cells.