Abstract: Railway is imperative for upgrading native industrial structure, transforming economic structure, strengthening foreign trade as well as enhancing the influence of great powers. As the middle route of Pan-Asian Railway system, China-Laos-Thailand Railway (CR) plays a significant role in promoting cross-border cooperation between China, Laos and Thailand as well as enhancing the construction of China-Indochina Peninsula Economic Corridor. Therefore, based on the regional development of natural barrier and non-barrier regions, this study chooses Yunnan Province in China, Laos and Thailand as the research area and selects 15 major cities along CR as the research objects. Based on the perspective of city flow network, by using Spatial Gravity model, Markov chain and Accessibility Analysis, the regional accessibility and the degree of inter-city linkages now were compared with those after the construction of CR. The results show that: 1) The construction of CR will make it easier to access natural barrier regions and promote the potential development ability in non-barrier regions. 2) The construction of CR will promote the regional accessibility of the study area comprehensively. Accessibility presents a pattern of decreasing radiation around CR. The improvements in accessibility, however, has little impact on natural barrier areas of Western Thailand and Eastern Laos. 3) The construction of CR will enhance the intensity of inter-city connection, realize the efficient reconfiguration of city flow network which grows from tree-like formation to lattice-like formation. The form of city network is more stable and mature. 4) In the process of network reconfiguration, the structure of city flow network is flatter and more dispersed, both the dispersion and dissipation of resources coexist, which makes the efficiency of inter-city linkages decrease.