Understanding the deformation mechanisms of rutile titanium dioxide (TiO2) nanowires (NWs) helps to improve the working reliability of TiO2-based nanoelectrical–mechanical systems and better apply strain engineering to these materials. This work investigated the bending deformation mechanisms at the atomic scale using in situ transmission electron microscopy (TEM) near room temperature. Large bending strains of 3.0% to 5.1% could be observed on individual TiO2 NWs near room temperature. The large bending deformation was attributed to the formation of rich stacking faults (SFs) lying on (101̅) planes and the nucleation and glide of extended dislocations belonging to the {101̅}⟨101⟩slip system.