TNF-alpha can stimulate a variety of kinases with the ability to activate non-muscle myosin II. As a result, increases in actin filament formation and actomyosin contractility (AMC) have been reported in response to TNF-alpha. These events are thought to play an important role in mediating TNF-alpha induced apoptosis but how they do so is unclear. In this study we prevented non-muscle myosin II activation in response to TNF-alpha by treating cells with the myosin light chain kinase (MLCK) inhibitor ML-7 or through isoform specific siRNA knockdown of myosin IIA and IIB. We found that treatment with ML-7 or knockdown of myosin IIB, but not IIA, impaired the cleavage of caspase 3 and caspase 8 as well as nuclear condensation in response to TNF-alpha. During this cell death process myosin II seemed to function independent of AMC since treatment of cells with blebbistatin or cytochalasin D failed to inhibit TNF-alpha induced caspase cleavage. Immunoprecipitation studies revealed associations of myosin IIB with clathrin and FADD in response to TNF-alpha suggesting a role for myosin IIB in TNFR1 endocytosis and DISC formation. Taken together these findings suggest that myosin IIB activation promotes TNF-alpha cell death signaling in a manner independent of its force generating property.