Updated publication reference for PubMed record(s): 30877198. Tunneling NanoTubes (TNTs) are membrane conduits that mediate long distance intercellular crosstalk in several organisms and play vital roles during development, pathogenic transmission and cancer metastasis. However, the molecular mechanisms of TNT formation and function remain poorly understood. The protein MSec is essential for TNT formation in multiple cell types. We identified a novel interaction of MSec with the endoplasmic reticulum chaperone ERp29. ERp29 depletion in mammalian cells led to a significant reduction in TNT formation, while its over-expression induced TNT formation, but strictly in an MSec-dependent manner. ERp29 stabilized MSec protein levels but not its mRNA levels, and the chaperone activity of ERp29 was required for maintaining MSec protein stability. Our study implicates MSec as a new target of ERp29 and reveals an indispensable role for the endoplasmic reticulum in the biogenesis of TNTs, thus suggesting new modalities for regulating TNT numbers.