Cytokinesis in eukaryotic cells is mediated by the contractile ring, an actomyosin-based structure which provides the force required to separate daughter cells. Isoforms of the actin-binding protein tropomyosin are also localised to the contractile ring in both fission yeast and human astrocytes. Although tropomyosin is required for cytokinesis in yeast, its precise role in the contractile ring is unknown. In this study we find that increased expression of a single tropomyosin isoform, tropomyosin 1, in U373MG astrocytoma cells leads to multinucleated cells and mitotic spindle defects. Furthermore, cells expressing increased levels of tropomyosin 1 usually fail to complete cytokinesis and this is accompanied by reduced accumulation of actin depolymerising factor/cofilin in the contractile ring. Adenovirus mediated expression of cofilin is able to relieve the tropomyosin 1 induced effects on cytokinesis. We conclude that tropomyosin 1 and cofilin play antagonistic roles within the contractile ring and that the balance between tropomyosin 1 and cofilin expression is important for cytokinesis.