Objective Tunneling nanotubes are membrane-tubule-like structures that exist between cells and have a direct long-distance biological information exchange function. Because the structure of TNTs is easily destroyed, exists for a short period of time and is unstable after formation, it is difficult to observe its dynamic formation and function. This study used the high content analysis system (HCA) combined with laser scanning confocal microscopy (LSCM) to try to observe the dynamic process of TNTs formation and its function of vesicular material transport.Methods Human lung adenocarcinoma A549 and cisplatin-resistant A549/DDP cells were labeled using fluorescent probes. Subsequently, HCA was used to observe the formation process of TNTs, LSCM to observe the three-dimensional structure of TNTs, and HCA combined with LSCM to analyze the vesicular material transport function of TNTs, respectively.Results TNTs structures can be formed between tumor cells of the same types (A549 or A549/DDP) or between different subtypes of tumor cells (A549 and A549/DDP). TNTs formed between A549/DDP cells were longer, thicker and had a higher formation index compared to A549 cells ( the length, diameter and formation index of TNTs for A549 and A549/DDP were 14.71 μm, 2.27 μm, 4 and 25.44 μm, 2.59 μm, 11, respectively). TNTs are formed in two steps: first, two cell cytoplasmic membranes first come into contact with each other and then fuse, and as the two cells translocate in opposite directions, the fused regions of the membranes are continuously elongated and narrowed, resulting in the formation of TNTs; second, two cells extend filopodia-like membrane projections and fuse when they come into contact with each other’s membrane projections, resulting in the formation of TNTs or one cell extends filopodia-like membrane projections and fuses after contacting the other cell membrane, thus forming TNTs. The transport of vesicles by TNTs is bidirectional. During transport, the rate and amounts of vesicles being transported vary depending on the stage of transport and the donor cell. The vesicular translocation from A549/DDP cells to A549 cells showed a fast initial rate and a slow terminal rate. A549 cells, as donor cells, transit vesicles to recipient A549/DDP cells in a higher number and proportion than A549/DDP as donor cells in the opposite direction.Conclusion The dynamic formation process and vesicular substance transport function of TNTs could be observed and analyzed by HCA combined with LSCM effectively.