Bladder cancer is the second most common urological cancer worldwide with low early diagnosis and high mortality. Since the time of diagnosis directly affects survival rate, early detection and precise biomarkers of bladder cancer are very important. Adenylate kinase 4 (AK4) is a key enzyme involved in cellular metabolism and multiple cancer development; however, the potential role of AK4 in bladder tumorigenesis is still unclear. Immunohistochemistry assay was conducted to evaluate the expression level of AK4 in 107 human bladder cancer tissues. Overall survival and recurrence-free survival were used to assess the prognosis of patients. Colony formation and MTT assays [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide] were performed to measure the proliferation capacity of tumor cells. Cell scratch assays and transwell assays were performed to measure the invasion capacity of tumor cells. The expression level of involved genes was measured by reverse transcription-polymerase chain reaction and western blot assays. The animal model was used to examine the effects of indicated protein on tumorigenesis and invasion in vivo. Herein, our study demonstrated that increased AK4 expression in patients with bladder cancer was associated with a poor prognosis. We further found that inhibition of AK4 in bladder cancer cell line T24 and 5637 can obviously inhibit the proliferation of cancer cells. Transwell assay results showed that down-regulated AK4 was related to the decreased metastasis of T24 and 5637 cells. In addition, AK4-shRNA transfected obviously inhibited tumor growth and metastasis in mice compared with the scramble group. Taken together, the results provide strong evidence of the involvement of AK4 in the progression of bladder cancer and suggest that it could have high potential as a therapeutic target of disease.