Based on the balanced panel data of 285 prefecture-level cities in China from 2011 to 2018, the impact of the opening of high-speed rail on the city's attraction of highly educated entrepreneurial talents was explored. The results show that the opening of high-speed rail significantly enhances the city's attractiveness to highly educated entrepreneurial talents. Mechanism analysis 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. It is 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. With the current new round of technological and industrial revolution advancing deeply, the construction of new types of transportation infrastructure represented by high-speed rail has become one of the core national strategies for promoting high-quality economic development in China. In this background, how local governments attract and gather high-quality human capital to drive regional innovation and economic growth has become an important issue that urgently needs to be explored.