Some Fe-based alloys containing Mn exhibit characteristic plastic deformation modes such as twin induced plasticity (TWIP) and transformation induced plasticity (TRIP). In this study, microstructural changes induced by a tensile test in polycrystalline Fe-25%Mn-0.6%C alloys exhibiting TWIP were characterized. Electron backscatter diffraction (EBSD) and two-dimensional (2D) X-ray diffraction (XRD) measurements using synchrotron radiation were carried out to characterize the microstructure and crystallographic orientation of the polycrystalline alloy samples. The samples were deformed by 10%, 30%, and 60% in a tensile test. The EBSD results showed that deformation twins appeared to form preferentially in grains with large Schmid factors for twinning, and these had an orientation of nearly <011> - <111> parallel to the tensile direction. In addition to twinning, plastic deformation by dislocation slip was also observed in the interior of grains and near grain boundaries. In the polycrystalline sample, the heterogeneous strain by twinning and dislocation slip evolved overall with tensile strain. The 2D-XRD results also indicated that dislocation slip as well as twinning occurs in tensile deformed samples, and the contribution of twinning and dislocation slip in grains depends on tensile strain. Therefore, multiple plastic deformation modes consisting of different deformation twins and dislocation slips are attributed to large elongation in Fe-Mn-C alloys.