Based on the balanced panel data of 285 prefecture-level cities in China from 2011 to 2018, this paper empirically analyzes the mechanism of the effect of the opening of high-speed rail and cities on the attraction of highly educated entrepreneurial talents. The results show that the opening of high-speed rail significantly enhances the city’s attractiveness to highly educated entrepreneurial talents. The mechanism test shows that the opening of high-speed rail significantly improves the level of capital environment, innovation and commodity factor flow in the city, and then improves the city’s attractiveness to highly educated entrepreneurial talents. Heterogeneity analysis shows that there is a significant positive correlation between the opening of high-speed rail in southern cities and the attraction of cities to entrepreneurial talents with college and bachelor’s degrees, the opening of high-speed rail in inland cities and the attraction of cities to entrepreneurial talents with college degrees, and the opening of high-speed rail in non-(new) first-tier cities and the attraction of cities to entrepreneurial talents with college degrees. The conclusions of this paper are helpful to comprehensively understand the micro mechanism and differential impact of urban high-speed rail on the flow of highly educated entrepreneurial talents, and provide references for the government to adjust the rational flow of human resources between regions.