Intercellular communication is mediated by fibrous structures known as tunneling nanotubes (TNTs). TNTs are present in diseased cells, including cancer cells, and facilitate the transport of intracellular substances crucial for cell survival. They contribute to cancer survival by enhancing metastatic and invasive abilities and are implicated in the drug resistance of cancer cells. However, previous studies have primarily focused on the biochemical signaling of TNTs, and their mechanical properties are largely unknown. We hypothesized that TNTs play a mechanical role in cell survival by regulating cell-cell distance, migration direction, and mechanical signaling between cells. In this study, we investigated the mechanical properties of TNTs in a human cervical cancer cell line, HeLa cells, through nanoindentation tests using atomic force microscopy (AFM). To minimize shape variation in the TNTs and enhance the efficiency of mechanical tests, the shape of the cells and the distance between neighboring cells were controlled using a microcontact printing method that regulates cell adhesion regions. By performing AFM indentation tests on the TNTs, we determined that their internal tension was 758.3 ± 372.5 pN, axial spring constant was 32.1 ± 14.2 pN/µm, and axial elastic modulus was 696.3 ± 578.0 kPa. Furthermore, we observed that the internal tension and axial elastic modulus correlated positively with TNT length, while the axial elastic modulus correlated negatively with TNT thickness; that is, the thinner and longer the TNTs, the stiffer and higher are their tension. This implies that longer TNTs can exert greater tension on neighboring cells, thereby facilitating the proliferation of cancer cells. These findings suggest that TNTs play a critical role in the mechanical communication of cancer cells and may influence various aspects of cell survival, significantly affecting the progression of cancer and other diseases.