The cytokine TRAIL is a promising cancer therapeutic candidate as it induces apoptosis selectively in transformed cells. TRAIL-induced clustering of its receptors (DR) is essential for the DISC complex formation, which induces cell death. The mechanism for TRAIL’s tumour selective effect is largely unknown. We identified the cytoskeleton proteins non-muscle myosin heavy chain IIa, IIb (NMHCIIa, NMHCIIb), myosin regulatory light chain (MLC2) and ß-actin as novel DR-interactors. An initially weak and TRAIL-induced abrogation of NMHCII/DR interaction correlated with efficient DISC formation in tumour cells. In contrast, a robust NMHCII/DR interaction that was sustained upon TRAIL stimulus was accompanied by incomplete DISC arrangement. Weakening the NMHCII/DR interaction in normal cells using chemical inhibitors enhanced TRAIL-induced apoptosis. Intriguingly, siRNA-mediated NMHCIIa- but not NMHCIIb depletion potently released TRAIL resistance in normal cells and influenced DISC composition. Reduced NMHCII/DR interaction in transformed cells was characterised by diminished MLC2 phosphorylation and altered protein expression of upstream regulatory kinases. Our results suggest that normal cell resistance to TRAIL-apoptosis is based on the interaction of cytoskeleton components with DR that is impaired upon transformation. Since NMHCII function in cell adhesion and migration, it will be interesting to study possible roles of the interaction in cell detachment and altered TRAIL sensitivity; moreover this link may provide clues as to the cause of TRAIL resistance in some cancers.