Improvements in passenger safety and fuel efficiency are crucial issues in the automotive industry. The use of advanced high-strength steel (AHSS) in automotive parts has been suggested as a solution to these issues because it enables large weight reduction and good crash worthiness. Strength and ductility are the key mechanical properties of automotive AHSS. However, high strength is often accompanied by low ductility, resulting in the so-called strength–ductility trade-off dilemma. Currently, there is an increasing demand for automotive AHSS that exhibits a balance between strength and ductility. Lightweight and high-strength medium manganese steel (MMnS with a Mn mass fraction of 3%–12%), as a representative example of the third-generation automotive AHSS, has an excellent combination of strength and plasticity due to the effective usage of the coupled transformation-induced plasticity (TRIP) effect and twinning-induced plasticity (TWIP) effect of the metastable austenite constituent upon deformation. To further improve comprehensive mechanical properties, MMnS with a high Mn content were developed to increase the austenite fraction. Thus, a duplex structure with an ultrafine ferrite and austenite matrix was formed. However, Mn segregation is likely to occur in MMnS with increasing Mn content, especially in the cases of Mn ＞10% (mass fraction), which considerably influences MMnS’s performance. Therefore, the effects of Mn. segregation on the overall mechanical properties, microstructure and deformation mechanism of MMnS need to be elucidated in more details. In this paper, the influence of Mn segregation on the microstructure and mechanical properties of MMnS with an austenite–ferrite duplex structure and a nominal composition of 0.3C–11Mn–2.7Al–1.8Si–Fe was systemically investigated. Specifically, the underlying mechanism of Mn segregation that affects the mechanical and microstructural behavior of cold-rolled and annealed MMnS was analyzed. The results show that Mn segregation causes the formation of a Mn-rich banded structure, where the grain size of austenite is larger, austenite stability is higher, and fine ferrite is distributed more sparsely on the austenite matrix compared with the case without Mn segregation. The dominant plastic deformation mechanism of austenite in the non-Mn segregation zone involves martensitic transformation and twinning, leading to the coupled TRIP + TWIP effect, while the rate of martensitic transformation is higher than those without Mn segregation. However, the martensitic transformation is inhibited in the austenite of the Mn-rich structure because of its higher stability, limiting the TRIP effect. Consequently, the test MMnS with Mn segregation shows lower ductility and fracture resistance than those without Mn segregation; moreover, its finer austenite enhances the work-hardening capacity.