Cytokinesis is a critical step of airway smooth muscle cell division that plays an essential role in the development and homeostasis of the respiratory system, as well as the progression of airway remodeling. The mechanisms that regulate smooth muscle cytokinesis are not fully understood. c-Abl (c-Abelson tyrosine kinase) is a nonreceptor protein tyrosine kinase that has a role in regulating actin dynamics and smooth muscle contraction. The role of c-Abl in cytokinesis has not been investigated. Here, c-Abl was found in the contractile ring, as evidenced by immunofluorescent microscopy. In addition, cortactin is a phosphorylatable protein that has been implicated in actin filament assembly. In this report, phosphorylated cortactin was also found in the contractile ring. Knockdown of c-Abl by RNA interference attenuated cortactin phosphorylation in the midzone and contractile ring formation. c-Abl knockdown decreased the number of cells undergoing cytokinesis, but increased the quantity of cells in metaphase/anaphase and the number of multinucleate cells. Treatment with the c-Abl pharmacological inhibitors, imatinib and GNF-5, had similar effects. Furthermore, the expression of a nonphosphorylatable cortactin mutant diminished cytokinesis. Finally, inhibition of actin filament assembly by latrunculin A attenuated c-Abl recruitment to the midzone. Thus, we propose a novel mechanism that regulates smooth muscle cell cytokinesis. c-Abl is recruited to the equator during cytokinesis, which may mediate cortactin phosphorylation. Phosphorylated cortactin may promote actin filament assembly, which facilitates contractile ring formation and cytokinesis. In addition, actin filament polymerization may facilitate the positioning of c-Abl to the contractile ring.