The deformation microstructure of single crystal superalloys CMSX-4 was investigated during uniaxial tensile with a strain rate of 5 × 10 −3 s −1 at room temperature. The objection of the current study is to evaluate the deformation twinning and investigate the deformation mechanism. Experimental results followed by transmission electron analysis show that deformation nanotwins and elemental segregation in single-crystal Ni-based superalloys were observed after tensile deformation at room temperature with a strain rate of 5 × 10 −3 s −1 . Furthermore, the relationship among deformation twinning, elemental segregation, and high local stress due to plastic deformation with increasing plastic strain was estimated and discussed. These findings provide valuable insights into understanding twinning and elemental segregation in the plastic deformation of single-crystal Ni-based superalloys. • Deformation nanotwins in CMSX-4 are initiated under tensile tests with a strain rate of 5 × 10 −3 s −1 at room temperature. • Element segregation can be observed at the twin zone at room temperature. • Deformation nanotwins in CMSX-4 is not necessarily accompanied by an atomic reordering process at room temperature.