Tunneling nanotubes (TNTs) are F-actin rich structures that connect distant cells, allowing the transport of many cellular components, including vesicles, organelles and different kind of molecules. TNTs are implicated in key cellular processes, such as development, immunity and tissue regeneration, but also in the transmission of various pathogens. Several molecular factors have been identified to participate in the regulation of TNT formation. One of the early molecular factors that is implicated in TNT formation is the exocyst complex. This complex is also involved in the tethering of secretory vesicles during secretion, which suggest that proteins that regulate vesicle trafficking could have a role in TNT formation. We have hypothesized that the formation of TNTs is modulated by proteins that participate in both, the regulation of vesicle trafficking and the remodelling of the actin cytoskeleton, and that these two processes are key for the formation of these structures. Since Rab GTPases are the major regulators of vesicle trafficking and also participate in actin cytoskeleton regulation, we examined the role of this protein family in TNT formation. First, we performed a screening of several different Rab proteins for its effect on TNT-dependent vesicle transfer. We found that Rab8a, Rab11a and Rab35 have a positive effect on vesicle transfer. Additional studies demonstrated that Rab8a and Rab11a overexpression also increase the number of TNT connected cells. Upon overexpression of VAMP3 (another protein involved in vesicle trafficking), we also observed an increase in the number of TNT connected cells. Further analysis showed that all three proteins, i.e. Rab11a, Rab8a and VAMP3, show an effect on TNT formation in a cascade dependent manner. To establish a relationship between Rab11a and Rab8a, we checked the role of Rabin8 (a protein that interacts with Rab11 and activates Rab8), on TNT formation and we found that it has no role in TNT formation. Additionally, we checked another protein whose function is similar to Rabin8, i.e. GRAB (guanine nucleotide exchange factor for Rab3A) and its role in TNT formation. The results show that GRAB overexpression increases TNT formation, but it acts in a pathway independent of Rab11 and Rab8a to regulate TNT formation. The analysis of Rab35, a protein involved in endocytic recycling, cytokinesis and neurite outgrowth, showed that the GTP-Rab35 bound form also increases TNT formation. Neurite outgrowth is an essential process in order to establish neural connectivity and vesicle recycling plays a crucial role in this process. Rab35 interacts with several proteins, that are involved in vesicle trafficking such as such as ACAP2 (acts as GAP of ARF6), MICAL-L1 (molecule interacting with CasL-like 1, which plays a role in vesicle recycling) EHD1 (a molecular scissor that has a role in vesicle scission). At the ARF6 positive endosomes, Rab35 recruits ACAP2 and MICAL-L1, and forms a complex that binds to EHD1 to regulate neurite outgrowth.Our data strongly suggest that these effectors may also be involved in the formation of TNTs. Individually, ACAP2, EHD1 and ARF6-GDP regulate TNT formation in a positive manner. But MICAL-L1 overexpression in cells shows no effect on TNTs. Also, preliminary data, indicates that Rab35 and EHD1 acts in a cascade mechanism to regulate TNT formation. This indicates that TNT formation and neurite outgrowth may act in a similar, but not exact pathway. The molecules identified here that have a role in TNT formation, constitute potential molecular targets for therapies aiming to block the spreading of pathogens that transfer through TNTs.This study proves that proteins that have a role in vesicle trafficking and neurite outgrowth, such as Rab proteins, are also involved in TNT formation.