Twinning structures and their interfacial segregation play a key role in strengthening of magnesium alloys. Micro-steps are frequently existed in the incoherent twin boundaries, while the effect of them on interface and interfacial segregation is still not clear. In this work, we performed an atomic-scale microstructure analysis using high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) to explore the effect of micro-steps on twin and its interfacial segregation in Mg-Ag alloy. Diffraction pattern of the incoherent {10 $\overline{1}$ 1} twin shows that the misorientation has a slight tilt of 5° from its theoretical angle of 125° due to the accumulated effects of the micro-steps and their misfit dislocations in twin boundaries. Most of the micro-steps in {10 $\overline{1}$ 1} twin boundary are in the height of 2 $d_{\left(10\overline{1}1\right)}$ and 4 $d_{\left(10\overline{1}1\right)}$ , respectively, and both of them have two types according to whether there are dislocations on the micro-steps. The twin boundary is interrupted by many micro-steps, which leads to a step-line distributed interfacial segregation. Moreover, the Ag tends to segregate to dislocation cores, which results in the interruption of interfacial segregation at the micro-steps with dislocations.