Tunnelling nanotubes (TNTs) are membranous connections that represent a unique type of intercellular communication in different cell types. They are associated with cell physiology and cancer pathology. The possible existence of tunnelling nanotubes communication between urothelial cancer and normal cells has not yet been elucidated. Therefore, we analyzed TNTs formed by T24 cells (human invasive cancer urothelial cells) and normal porcine urothelial (NPU) cells, which serve as surrogate models for healthy human urothelial cells. Monocultures and cocultures of NPU and T24 cells were established and analyzed using live-cell imaging, optical tweezers, fluorescence microscopy, and scanning electron microscopy. TNTs of NPU cells differed significantly from tunnelling nanotubes of T24 cells in number, length, diameter, lipid composition, and elastic properties. Membrane domains enriched in cholesterol/sphingomyelin were present in tunnelling nanotubes of T24 cells but not in NPU cells. The tunnelling nanotubes in T24 cells were also easier to bend than the tunnelling nanotubes in NPU cells. The tunnelling nanotubes of both cell types were predominantly tricytoskeletal, and contained actin filaments, intermediate filaments, and microtubules, as well as the motor proteins myosin Va, dynein, and kinesin 5B. Mitochondria were transported within tunnelling nanotubes in living cells, and were colocalized with microtubules and the microtubule-associated protein dynamin 2. In cocultures, heterocellular tunnelling nanotubes were formed between NPU cells and T24 cells and vice versa. The presence of connexin 43 at the end of urothelial tunnelling nanotubes suggests a junctional connection and the involvement of tunnelling nanotube in signal transduction. In this study, we established a novel urothelial cancer-normal coculture model and showed cells in the minority tend to form tunnelling nanotubes with cells in the majority. The condition with cancer cells in the minority is an attractive model to mimic the situation after surgical resection with remaining cancer cells and may help to understand cancer progression and recurrence. Our results shed light on the biological activity of tunnelling nanotubes and have the potential to advance the search for anticancer drugs that target tunnelling nanotubes.