Based on the panel data of 283 cities above prefecture level in China from 2004 to 2016, this paper uses the differential regression model to analyze the impact of the opening of high-speed rail on population mobility and its related “flow economy” agglomeration level. Overall, the opening of high-speed rail has promoted the population flow and the agglomeration of “flow economy” in cities along the line. This impact is heterogeneous due to the differences in regional economic development level of high-speed rail cities. With the passage of time, the agglomeration level of “flow economy” in cities with early high-speed rail opening changed slightly in an inverted U-shape. In the supporting construction of high-speed rail network, we should not only pay attention to the spatial balanced investment and realize the planning and guiding role of high-speed rail network in reshaping the economic and geographical pattern and rational population distribution, but also pay attention to the siphon effect of central cities caused by the opening of high-speed rail in the short term, so as to avoid inefficient investment.