The high-speed railway (HSR) has been generally regarded as a valid enhancement to national accessibility. However, the debate on the impact of HSR on regions has not reached a consensus. Therefore, we create the accessibility model in nine provinces along the Yellow River based on different years, which incorporates urban scale, travel time, and service frequency of HSR, to dynamically analyze the spatial pattern of urban agglomeration. Moreover, we adapt the Difference-in-differences model to explore the impact on the regional and urban economy and population. The results indicate that HSR significantly improves the accessibility level of most cities in the east and south of nine provinces along the Yellow River, showing a significant “corridor effect.” In terms of urban development potential, the spatial distribution pattern of “mass-shaped high-value areas in the east and point-shaped high-value areas in the west” is observed. Furthermore, empirical results show a greater agglomeration rather than diffusion, large cities benefit from HSR with economic promotion, however, HSR causes population loss and economic downturn in small and medium-sized cities. The paper highlights that the HSR does not always bring growth opportunities, a coordinated policy should be implemented for more integrated development of future HSR planning and construction.