Urinary bladder urothelial carcinoma (UBUC) encompasses about 90% of all bladder cancer cases, and the mainstream treatment is the transurethral resection of the bladder tumor followed by intravesical instillation. High rates of mortality, recurrence, and progression in bladder cancer have stimulated the search for alternative adjuvant therapies. The aim of this study was to investigate the potential of melatonin as adjuvant therapy in bladder cancer. Cell viability and clonogenic ability were assessed by an MTT assay and colony formation. Cell cycle and apoptosis analysis were performed by flow cytometry and Hoechst 33342 staining, while cell metastasis capacity was measured by wound healing and transwell assays. Potential mechanisms were investigated by an oncology array and verified via western blotting. The melatonin treatment significantly reduced T24 and UMUC3 bladder cancer cell proliferation and clonogenic ability. G1 arrest and sub-G1 accumulation in the T24 and UMUC3 cells led to cell proliferation suppression and cell death, and Hoechst 33342 staining further verified the apoptosis induction directly by melatonin. Moreover, melatonin weakened cell motility and invasiveness. Based on the oncology array results, we demonstrated that melatonin exerts its anti-cancer effect by down-regulating the HIF-1α and NF-κB pathways and downstream pathways, including Bcl-2, leading to cell cycle arrest and apoptosis induction in the UBUC cells. Overall, these findings support the potential of melatonin as adjuvant therapy in bladder cancer.