The expression of microRNA‑335 (miR‑335) has been demonstrated to be downregulated in numerous types of cancer. Thus far, no previous studies have investigated the miR‑335 expression in bladder cancer. In the present study, the expression and effects of miR‑335 were assessed in bladder cancer. The results of the present study provided, to the best of our knowledge, the first evidence that miR‑335 is downregulated in the tumor tissue of patients with bladder cancer. Following transfection of miR‑335, MTT, cell migration and invasion, luciferase and western blot assays were conducted in bladder cancer cell lines. The results demonstrated that miR‑335 inhibited cell proliferation, migration and invasion in T24 and EJ cells. In addition, the results suggested that miR‑335 directly targets Rho‑associated protein kinase 1 (ROCK1) in bladder cancer. The present study provided a novel therapeutic target, the miR‑335/ROCK1 axis in bladder cancer. The suggested approach will be beneficial in developing an effective treatment against bladder cancer.