Muscle invasive bladder cancer is an aggressive type of epithelial tumor with a high rate of metastasis. For bladder cancer cells to reach the muscle layer, cells must invade through an urothelial cell monolayer (transurothelial invasion) and basement membrane. However, the process by which transurothelial invasion occurs has not been fully characterized. In this study we developed a novel method to evaluate the transurothelial invasion capacity and investigated its cellular and molecular processes using primary culture cells from bladder cancer patients. The analysis revealed that compared with the prognosis for patients with non‑muscle invasive bladder cancer that of patients with muscle invasive bladder cancer was particularly poor due to metastatic recurrence. Cancer cells from patients with muscle invasive bladder cancer exhibited a higher invasive capacity through the urothelial cell monolayer compared with those from non‑invasive bladder cancer patients. Furthermore, muscle invasive bladder cancer cells demonstrated a greater ability to form invadopodia, the filamentous actin‑based membrane protrusions required for matrix degradation and invasion compared with non‑invasive cells. Bladder cancer cell lines were established with reduced invadopodia formation by silencing the expression of cortactin, an essential component of invadopodia. The cortactin knockdown bladder cancer cells with reduced invadopodia formation demonstrated a markedly reduced ability to invade through the urothelial cell monolayer, indicating that invadopodia are essential for transurothelial invasion. The results indicate that invadopodia formation is required for muscle invasion of aggressive bladder cancer cells.